Morphology,toxicity, and phylogeny of Alexandrium (Dinophyceae) species along the coast of China

The toxigenic genus Alexandrium includes ∼30 species, but information about its biogeography at a regional scale is limited. In this study, we explored the diversity of Alexandrium along the coast of China by incubating resting cysts collected from 7 sites. A total of 231 strains of Alexandrium belonging to 7 morphospecies were found. Among them, Alexandrium andersonii, Alexandrium fraterculum, Alexandrium leei, Alexandrium pseudogonyaulax, and Alexandrium tamutum were recorded from the China Sea for the first time. Partial large subunit (LSU) and/or internal transcribed spacer region (ITS1, ITS2, and 5.8S rDNA) sequences revealed two ribotypes of Alexandrium andersonii, Alexandrium leei, and Alexandrium tamarense: Atama complex Group I and IV. Atama complex Group I was exclusively distributed in the Yellow Sea and the Bohai Sea, whereas Group IV was restricted to the East China Sea and South China Sea. Atama complex Group I produced mainly N-sulfocarbamoyl toxins (C1/C2, 61–79% of total toxins) and gonyautoxins (GTX1/4, 17–37%). Alexandrium ostenfeldii strain ASBH01 produced NEO and STX exclusively (65% and 35%, respectively). Our results support the premise that Atama complex Group I is endemic to the Asian Pacific and includes cold water species, whereas Atama complex Group IV tends to inhabit warmer waters.     

First Records of Dinoflagellates Alexandrium margalefi Balech, 1994 and A. tamutum Montresor, Beran et John, 2004 in the Seas of the Russian Far East

New records of two Alexandrium species are reported for the Russian seas. A. margalefi Balech was found in Peter the Great Bay (Sea of Japan), and A. tamutum Montresor, Beran et John was found in Aniva and Sakhalinskii bays (Sea of Okhotsk). Both species were observed in summer in small numbers at water temperatures of 22– 24°C and 8.6–11°C, respectively. Data on the morphology and distribution of the species is provided.Original Russian Text Copyright © 2005 by Biologiya Morya, Selina, Morozova.     

Resting cysts,and effects of temperature and salinity on the growth of vegetative cells of the potentially harmful species Alexandrium insuetum Balech (Dinophyceae)

The potentially harmful species Alexandrium insuetum established by the incubation of resting cysts isolated from sediment trap samples collected at Jinhae-Masan Bay, Korea was characterized by morphological and phylogenetic analysis. The effects of temperature and salinity on the growth of A. insuetum were also investigated. The resting cysts are characterized by a spherical shape, a small size (20–25 μm) and the presence of either three or four red accumulation bodies. The similarity of morphological features of the resting cysts to those of other species of the minutum group (consisting of Alexandrium minutum and A. tamutum) indicates that the morphological features of resting cysts might improve the accuracy of the grouping of Alexandrium species. A. insuetum germinated from the resting cysts is morphologically consistent with vegetative cells reported from Korean and Japanese coastal areas, and has an partial large subunit (LSU) rDNA sequence identical to that from Japanese strains. The growth of A. insuetum was observed between salinity 20 and 35, with increasing temperature; however at 25 °C, A. insuetum could grow even at the salinity of 15. The highest growth rate (0.60 d⁻¹) was observed at 25 °C and the salinity of 25, which is higher than the previously reported growth rate of A. tamarense, which is responsible for outbreaks of paralytic shellfish poisoining and blooms in Jinhae-Masan Bay. These results suggest that the proliferation of A. insuetum in Jinhae-Masan Bay is likely to be highest during the summer.     

The species composition and quantitative distribution of the diatom genus Pseudo-nitzschia H. Peragallo, 1990 in Russian waters of the Sea of Japan and the Sea of Okhotsk

This paper studies the species composition and quantitative distribution of diatoms that belong to the genus Pseudo-nitzschia in the Russian waters of the Sea of Japan and the Sea of Okhotsk. In total, 11 species of this genus were found in the area, including 7 that are known as being potentially toxic. The highest concentrations of Pseudo-nitzschia microalgae (1.4 × 10⁶–2.7 × 10⁶ cells/L) were found in the summer and autumn in the Peter the Great Bay of the Sea of Japan and the lowest concentrations (2.5 × 10²–1 × 10⁴ cells/L) were found in the Sakhalinsky and Akademiya bays of the Sea of Okhotsk. The species diversity of potentially toxic diatoms was greatest (seven species) and the cell concentrations highest (over 6 × 10⁵ cells/L) in the Peter the Great Bay, Sea of Japan, and in the Aniva Bay, Sea of Okhotsk. The density of potentially toxic species was highest near the northeastern coast of Sakhalin Island, in the Amur River estuary, and in adjacent waters. This paper also presents geographical distribution maps of Pseudo-nitzschia species and maps of the density distribution of potentially toxic microalgae over the studied area and identifies potential amnesic shellfish poisoning areas.     

Dinoflagellate cyst records in recent sediments from Daya Bay, South China Sea

Nine sediment cores of 8–26 cm in length were collected from two basins of Daya Bay, the South China Sea, by Tokyo University Fisheries Oceanography Laboratory core sampler in August 2001 to investigate the distribution of dinoflagellate resting cysts. In the present study, 51 different cyst morphotypes representing 22 genera were identified from 65 sediment samples. Among them, there were 21 autotrophic species and 30 heterotrophic ones. Cyst species richness in each sample varied from 12 to 29, while the values of Shannon‐Weaver diversity index (H′) were between 0.15 and 4.13. There were an obvious increase in both species richness and values of H′in 2–6 cm sediments. Cyst concentrations varied from 154 to 113 483 cysts per gram dry weight sediment, and were much higher in upper sediments. Scrippsiella trochoidea was the most dominant cyst type, which took up over 90% of cyst assemblages in the upper sediments. The abrupt increase of S. trochoidea cysts in the surface sediments reflected the bloom of this species in Daya Bay in 2000. The results from cyst assemblages showed some trend of changes in water quality in this area, and indicated a typical type of pollution caused by cultural eutrophication, which started in the 1980s and greatly accelerated in the middle of 1990s. Cysts of Alexandrium, mainly those of Alexandrium catenella and Alexandrium tamarense complex, occurred frequently and abundantly in this area, with the highest concentration and relative frequency of 503 cysts per gram dry weight sediment and 22.3%, respectively. The high abundance of Alexandrium cysts provided rich ‘seed bed’ for Alexandrium blooms and was also an important source of paralytic shellfish poisoning toxins, especially in winter.     

Dinoflagellate cysts in recent marine sediments of the western coast of the Bering Sea

The quantitative and qualitative composition of live dinoflagellate cysts was studied in the upper two-centimeter layer of recent marine sediments that were collected at 19 stations in the coastal waters of the western Bering Sea. A total of 28 types of identified cysts belonged to the following 11 genera: Alexandrium, Diplopsalis, Ensiculifera, Gonyaulax, Gyrodinium, Pentapharsodinium, Polykrikos, Preperidinium, Protocera- tium, Protoperidinium, and Scrippsiella. The morphology of dinoflagellate cysts from recent sediments of Russian seas, such as the shape, the size, and also the structure of the phragma, including the processes and the archeopyle, was described comprehensively for the first time. Cysts of the species Gonyaulax spinifera, Pentapharsodinium dalei, Protoceratium reticulatum, Protoperidinium americanum, P. conicoides, P. subinerme, Scrippsiella crystallina, and S. trochoidea were the most widespread. Those of the potentially toxic species Alexandrium tamarense were also widely distributed and prevailed in the studied area. Their concentration varied from 0 to 25 860 cells/cm³; the maximum concentration was recorded in Pavel Bay, Koryak Okrug, and Kamchatka.     

Resting stages of microalgae in recent marine sediments of Peter the Great Bay,Sea of Japan

Data on the qualitative and quantitative composition of resting stages of planktonic microalgae in recent marine sediments of Peter the Great Bay (Sea of Japan) over the period 2000–2007 are presented. A total of sixty one morphological forms of resting stages represented by dinoflagellate and raphidophyte cysts and diatom spores and resting cells were recorded in the sediment samples. This study revealed cysts of the potentially toxic species Alexandrium tamarense, A. cf. minutum, Alexandrium sp., Gymnodinium catenatum (PSP toxin producers), and Protoceratium reticulatum (yessotoxin producer); resting cells of Pseudo-nitzschia sp. (potential producer of domoic acid); and cysts of bloom-forming species Cochlodinium cf. polykrikoides and Heterosigma cf. akashiwo.     

Abundance and distribution of toxic Alexandrium tamarense resting cysts in the sediments of the Chukchi Sea and the eastern Bering Sea

Abundance and distribution of the toxic dinoflagellate Alexandrium tamarense species complex resting cyst were investigated in the eastern Bering Sea and the Chukchi Sea for the first time. Sediment samples (top 0–3 cm depth) were collected from the continental shelf of the eastern Bering Sea (17 stations) and the Chukchi Sea (13 stations) together with a long core sample (top 0–21 cm depth) from one station in the Chukchi Sea during 2009–2012. The cysts were enumerated using the primuline staining method. Species identification of the cysts was carried out with multiplex PCR assay and the plate morphology of vegetative cells germinated from cysts in the both areas. Alexandrium cysts were widely detected in the both areas, ranging from not detected (<1 cysts cm⁻³) to 835 cysts cm⁻³ wet sediment in the eastern Bering Sea and from not detected (<1 cysts cm⁻³) to 10,600 cysts cm⁻³ in the Chukchi Sea, and all isolated cysts were genetically and morphologically identified as the North American clade A. tamarense. Their cysts were mainly distributed in the shallow continental shelf where the water depth was less than 100 m in both areas. The cysts were detected from the deep layer (18–21 cm depth of sediment core) of the long core sample. The present study confirmed the abundant existence of A. tamarense with wide range of distribution in these areas. This fact suggests that A. tamarense vegetative cells have appeared in the water column in the both areas. Furthermore, these abundant cyst depositions indicate that this species originally distributed in the Arctic and subarctic regions and well adapted to the environments in the marginal ice zone.     

Vertical distribution and germination ability of Alexandrium spp. cysts (Dinophyceae) in the sediments collected from Kure Bay of the Seto Inland Sea, Japan

The vertical distribution of Alexandrium tamarense/ catenella (hereinafter Alexandrium spp.) cysts was investigated with special attention to living cysts filled with fresh protoplasm and empty cysts. In addition, based on the incubation experiments of Alexandrium spp. cysts, the germination ability of the cysts was examined. A sediment core 63 cm in length, collected from Kure Bay of the Seto Inland Sea, West Japan, in September 2000, was provided for an analysis on the vertical distribution of Alexandrium spp. cysts. Samples from every 1 cm interval depth from the top down to 13 cm depth of the same core were taken to examine the germination ability of the cysts. Results show that Alexandrium spp. cysts were continuously observed from 59 to 60 cm depth to the top. The cyst densities in the upper parts of the core (from 9 to 10 cm depth to the top) were much more abundant those that in the lower parts (below 10–11 cm depth). The relationship between living and empty cysts in each depth did not reveal a positive correlation with the sediment depth. Based on the sedimentation rate of the core sediment (approximately 1.6 cm/year), Alexandrium spp. cysts have been produced since 1962, and a remarkable increase of these cysts was observed from ca 1993. Such a rapid increase of Alexandrium spp. cysts has probably been as a result of dense blooms of A. tamarense occurring since 1992 in Hiroshima Bay, including Kure Bay. The germination of Alexandrium spp. cysts was observed in samples collected from the top to 12–13 cm depth of the core. It suggests that the Alexandrium spp. cysts can keep the germination ability for more than 8 years.     

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 coastal waters 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.     

Possible spreading of toxic Alexandrium tamarense blooms on the Chukchi Sea shelf with the inflow of Pacific summer water due to climatic warming

A high abundance of resting cysts of the toxic dinoflagellate Alexandrium tamarense was recently reported in the vast continental shelf of the Chukchi Sea in the Arctic Ocean, suggesting that the species is widespread in the shelf. Nevertheless, little is known about the occurrence of A. tamarense vegetative cells in the water column of the arctic. Sea ice reduction and the inflow of Pacific summer water (PSW) through the Bering Strait have recently increased owing to warming in the shelf. To determine the spatial and temporal distributions of A. tamarense in the Chukchi Sea shelf and their relationship to the inflow of PSW, field samplings were conducted in the Chukchi Sea and north Bering Sea shelves three times during the summer of 2013 from July to October. Vegetative cells of A. tamarense was detected in both shelves at all sampling periods with a maximum density of 3.55 × 10³ cells L⁻¹. This species was also observed at the station at 73°N, indicating the northernmost record of this species to date. The center of the A. tamarense distribution was between the north Bering and south Chukchi Sea shelf during the first collection period, and spread to the north Chukchi Sea shelf during the second and third collection periods. The species occurrences were mainly observed at stations affected by the PSW, especially Bering shelf water. Water structure of PSW was characterized by warmer surface and bottom water temperatures, and increased temperatures may have promoted the cell growth and cyst germination of A. tamarense. Therefore, it is suggested that an increase in the PSW inflow owing to warming promotes toxic A. tamarense occurrences on the Chukchi Sea shelf.     

A genetic analysis of the beluga whale Delphinapterus leucas (Cetacea: Monodontidae) from summer aggregations in the Russian Far East

The genetic structure of four summer aggregations of the Beluga Whale, Delphinapterus leucas, in Sakhalin Bay and Udskaya Bay, off the western coast of Kamchatka in the Sea of Okhotsk and in the Anadyr Estuary of the Bering Sea was analyzed through nucleotide sequencing of the mtDNA control region and detection of the allelic composition of nine microsatellite loci in nuclear DNA. It has been shown that each of the aggregations features a unique set of maternal lines, which indicates a high degree of philopatry in this species. Beluga whales of the Anadyr Estuary are genetically isolated from those of the Sea of Okhotsk. Beluga whales of the summer aggregations of Sakhalin Bay and those from Udskaya Bay share a common gene pool and belong to a single population, while the whales that summer off western Kamchatka with great consistency may be attributed to a different population. Comparison of nucleotide sequences of the mtDNA in beluga whales from various waters of the Russian Far East and North America allowed us to propose a hypothesis about how the structure of beluga whale populations formed in the North Pacific during the postglacial period.     

Recurrent vernal presence of the toxic Alexandrium tamarense/Alexandrium fundyense (Dinoflagellata) species complex in Narragansett Bay,USA

The vernal occurrence of toxic dinoflagellates in the Alexandrium tamarense/Alexandrium fundyense species complex in an enclosed embayment of Narragansett Bay (Wickford Cove, Rhode Island) was documented during 2005 and 2009–2012. This is the first report of regular appearance of the Alexandrium fundyense/Alexandrium tamarense species complex in Narragansett Bay. Thecal plate analysis of clonal isolates using SEM revealed cells morphologically consistent with both Alexandrium tamarense Lebour (Balech) and Alexandrium fundyense Balech. Additionally, molecular analyses confirmed that the partial sequences for 18S through the D1–D2 region of 28S were consistent with the identity of the two Alexandrium species. Toxin analyses revealed the presence of a suite of toxins (C1/2, B1 (GTX-5), STX, GTX-2/3. Neo, and GTX-1/4) in both Alexandrium tamarense (6.31 fmol cell⁻¹ STX equiv.) and Alexandrium fundyense (9.56 fmol cell⁻¹ STX equiv.) isolated from Wickford Cove; the toxicity of a Narragansett Bay Alexandrium peruvianum isolate (1.79 fmol cell⁻¹ STX equiv.) was also determined. Combined Alexandrium tamarense/Alexandrium fundyense abundance in Wickford Cove reached a peak abundance of 1280 cells L⁻¹ (May of 2010), with the combined abundance routinely exceeding levels leading to shellfishing closures in other systems. The toxic Alexandrium tamarense/Alexandrium fundyense species complex appears to be a regular component of the lower Narragansett Bay phytoplankton community, either newly emergent or previously overlooked by extant monitoring programs.     

Changes in abundances of Alexandrium tamarense resting cysts after the tsunami caused by the Great East Japan Earthquake in Funka Bay,Hokkaido, Japan

The 2011 Great East Japan Earthquake and the subsequent huge tsunami greatly affected both human activity and the coastal marine ecosystem along the Pacific coast of Japan. The tsunami also reached Funka Bay in northern Japan and caused serious damage to the scallop cultures there, and this tsunami was believed to have affected the coastal environments in the bay. Therefore, we investigated the changes in the spatial abundance and distribution of the toxic dinoflagellates Alexandrium tamarense cysts before the tsunami (August 2010) and after the tsunami (May 2011, August 2011, May 2012 and August 2012) in the bay. Further, monthly sampling was conducted after the tsunami to identify seasonal changes of Alexandrium catenella/tamarense cysts and vegetative cells. Significant increases were observed in the populations of A. catenella/tamarense cysts, comparing the abundances before the tsunami (in August 2010; 70 ± 61 cysts g⁻¹ wet sediment) to those just after it (in May 2011; 108 ± 84 cysts g⁻¹ wet sediment), and both A. tamarense bloom (a maximum density was 1.3 × 10³ cells L⁻¹) and PSP (Paralytic Shellfish Poisoning) toxin contamination of scallops (9.4 mouse unit g⁻¹ was recorded) occurred in the bay. Seasonal sampling also revealed that the encystment of A. tamarense and the supply of the cysts to bottom sediments did not occur in the bay from September to April. These results strongly suggested that the mixing of the bottom sediments by the tsunami caused the accumulation of the toxic A. tamarense cysts in the surface of bottom sediment through the process of redeposition in Funka Bay. Moreover, this cyst deposition may have contributed to the toxic bloom formation as a seed population in the spring of 2011.     

Comparative studies on morphology, ITS sequence and protein profile of Alexandrium tamarense and A. catenella isolated from the China Sea

The Alexandrium tamarense species complex is a closely related cosmopolitan toxigenic group of morphology-based species, including A. tamarense, A. catenella and A. fundyense. This study investigated the morphology, internal transcribed spacer (ITS) sequence and protein profile of A. tamarense and A. catenella grown in the same culture conditions using a combination of scanning electronic microscope (SEM), molecular and proteomic approaches. The results showed that all Alexandrium strains had the plate formula of Po, 4′, 6″, 6C, 8S, 5″′, 2″″. The ventral pore, a key conventional morphological feature to discriminate A. tamarense and A. catenella, was usually present in the first apical plate of ten A. tamarense strains, however, it was found to be absent in some cells of one Alexandrium strain, ATGX01. A. tamarense and A. catenella shared an identical ITS sequence with a minor variation at intraspecific level. Protein profiles of A. catenella DH01 and A. tamarense DH01, isolated from the same region of the East China Sea, showed no significant difference, the similarity of protein profiles of the two species reached 99% with a few proteins unique to one or the other. The present results suggest that the ventral pore is not a consistent morphological feature in the Alexandrium genus, and that A. tamarense and A. catenella are conspecific and should be redesignated to one species.     

Development of Molecular Identification Method for Genus Alexandrium (Dinophyceae) Using Whole-Cell FISH

We have developed a method to identify species in the genus Alexandrium using whole-cell fluorescent in situ hybridization with FITC-labeled oligonucleotide probes that target large subunit ribosomal rRNA molecules. The probes were designed based on the sequence of the rDNA D1-D2 region of Alexandrium species. DNA probes specific for toxic A. tamarense and A. catenella and nontoxic A. affine, A. fraterculus, A. insuetum, and A. pseudogonyaulax, respectively, were applied to vegetative cells of all above Alexandrium species to test the sensitivity of the probes. Each DNA probe hybridized specifically with vegetative cells of the corresponding Alexandrium species and showed no cross-reactivity to noncorresponding Alexandrium species. In addition, no cross-reactivity of the probes was observed in experiments using concentrated natural seawater samples. The TAMAD2 probe, which is highly specific to A. tamarense, a common toxic species in Korean coastal waters, provides a simple and reliable molecular tool for identification of toxic Alexandrium species.     

Experimental study on the impact of dinoflagellate Alexandrium species on populations of the rotifer Brachionus plicatilis

To investigate harmful effects of the dinoflagellate Alexandrium species on microzooplankton, the rotifer Brachionus plicatilis was chosen as an assay species, and tested with 10 strains of Alexandrium including one known non-PSP-producer (Alexandrium tamarense, AT-6). HPLC analysis confirmed the PSP-content of the various strains: Alexandrium lusitanicum, Alexandrium minutum and Alexandrium tamarense (ATHK, AT5-1, AT5-3, ATCI02, ATCI03) used in the experiment were PSP-producers. No PSP toxins were detected in the strains Alexandrium sp1, Alexandrium sp2.Exposing rotifer populations to the densities of 2000 cells ml⁻¹ of each of these 10 Alexandrium strains revealed that the (non-PSP) A. tamarense (AT-6) and two other PSP-producing algae: A. lusitanicum, A. minutum, did not appear to adversely impact rotifer populations. Rotifers exposed to these three strains were able to maintain their population numbers, and in some cases, increase them. Although some increases in rotifer population growth following exposures to these three algal species were noted, the rate was less than for the non-exposed control rotifer groups.In contrast, the remaining seven algal strains (A. tamarense ATHK, AT5-1, AT5-3, ATCI02, ATCI03; also Alexandrium sp1 and Alexandrium sp2) all have adverse effects on the rotifers. Dosing rotifers with respective algal cell densities of 2000 cells ml⁻¹ each, for Alexandrium sp1, Alexandrium sp2, and A. tamarense strains ATHK and ATCI03 showed mean lethal time (LT₅₀) on rotifer populations of 21, 28, 29, and 36h, respectively. The remaining three species (A. tamarense strains AT5-1, AT5-3, ATCI02) caused respective mean rotifer LT₅₀s of 56, 56, and 71 h, compared to 160 h for the unexposed “starved control” rotifers. Experiments to determine ingestion rates for the rotifers, based on changes in their Chlorophyll a content, showed that the rotifers could feed on A. lusitanicum, A. minutum and A. tamarense strain AT-6, but could graze to little or no extent upon algal cells of the other seven strains. The effects on rotifers exposed to different cell densities, fractions, and growth phases of A. tamarense algal culture were respectively compared. It was found that only the whole algal cells had lethal effects, with strongest impact being shown by the early exponential growth phase of A. tamarense. The results indicate that some toxic mechanism(s), other than PSP and present in whole algal cells, might be responsible for the adverse effects on the exposed rotifers.     

RESTRICTION FRAGMENT LENGTH POLYMORPHISM OF RIBOSOMAL DNA INTERNAL TRANSCRIBED SPACER AND 5.8S REGIONS IN JAPANESE ALEXANDRIUM SPECIES (DINOPHYCEAE)1

The 5.8S ribosomal RNA (rDNA) gene and flanking internal transcribed spacers (ITS1 and ITS2)from 9 isolates of Alexandrium catenella (Whedon and Kofoid) Taylor, 11 isolates of A. tamarense (Lebour) Taylor, and single isolates of A. affine (Inoue et Fukuyo) Balech, A. insuetum Balech, and A. pseudogonyaulax (Biecheler) Horiguchi ex Yuki et Fukuyo comb. nov. from various locations in Japan were amplified using the polymerase chain reaction (PCR) and subjected to restriction fragment-length polymorphism (RFLP) analysis. PCR products from all strains were approximately 610 bp, inclusive of a limited region of the 18S and 28S rRNA coding regions. RFLP analysis using four restriction enzymes revealed six distinct classes of rDNA (“ITS types”). Restriction patterns of A. catenella were uniform at the intra-specific level and clearly distinguishable from those of A. tamarense. The patterns associated with A. tamarense (“tamarense group”) were also uniform except for one strain, WKS-1. Some restriction fragments from WKS-1 were in common with those of A. catenella or A. tamarense, whereas some were distinct from all Alexandrium species tested. Alexandrium affine, A. insuetum, and A. pseudogonyaulax carry unique ITS types. The ITSs of the “tamarense group” exhibit sequence heterogeneity. In contrast, the ITSs of all other isolates (including WKS-1) appear homogeneous. RFLP analysis of the 5.8S rDNA and flanking ITSs regions from Alexandrium species reveals useful taxonomic and genetic markers at the species and/or population levels.