NITROGENOUS NUTRITION OF ALEXANDRIUM CATENELLA (DINOPHYCEAE) IN CULTURES AND IN THAU LAGOON,SOUTHERN FRANCE1

Alexandrium catenella (Whedon et Kofoid) Balech was isolated from Thau lagoon (northern Mediterranean) and its growth and uptake characteristics measured for nitrate, ammonium, and urea. Although affinity constants did not indicate a preference for ammonium over nitrate, there was a strong inhibition of nitrate uptake by ammonium when both nitrogen (N) sources were present. Nitrogen budgets during growth in cultures revealed major imbalances between decreases in dissolved N and increases in particulate N, indicating excretion of dissolved organic N during the early part of the growth phase and uptake during the later part. A quasi‐unialgal bloom in November 2001 (4×10⁶ cells·L^?1) allowed measurements of uptake of nitrate, nitrite, ammonium, and urea; net and gross growth rate of A. catenella; and grazing rates on this organism. The affinity constants indicate that it is not a strong competitor for the N nutrients tested when these are in low concentrations (<10 μgat N·L^?1), compared with other members of the phytoplankton community. Indirect evidence from cultures indicate that dissolved organic N compounds could be important in triggering those blooms. Finally, the strongly unbalanced growth observed in the field indicates that A. catenella exhibits a storage rather than a growth response to a nutrient pulse and is adapted to low frequency events such as the passage of frontal disturbances. The disappearance of A. catenella was due to grazing that balanced growth at the peak of the bloom.     

GROWTH AND PHOSPHORUS UPTAKE BY THE TOXIC DINOFLAGELLATE ALEXANDRIUM CATENELLA (DINOPHYCEAE) IN RESPONSE TO PHOSPHATE LIMITATION1

Alexandrium catenella (Whedon et Kof.) Balech has exhibited seasonal recurrent blooms in the Thau lagoon (South of France) since first reported in 1995. Its appearance followed a strong decrease (90%) in phosphate (PO₄^3?) concentrations in this environment over the 1970–1995 period. To determine if this dinoflagellate species has a competitive advantage in PO₄^3?‐limited conditions in terms of nutrient acquisition, semicontinuous cultures were carried out to characterize phosphorus (P) uptake by A. catenella cells along a P‐limitation gradient using different dilution rates (DRs). Use of both inorganic and organic P was investigated from measurements of ³³PO₄^3? uptake and alkaline phosphatase activity (APA), respectively. P status was estimated from cellular P and carbon contents (Q_P and Q_C). Shifts in trends of Q_P/Q_C and Q_P per cell (Q_P·cell?1) along the DR gradient allowed the definition of successive P‐stress thresholds for A. catenella cells. The maximal uptake rate of ³³PO₄^3? increased strongly with the decrease in DR and the decrease in Q_P/Q_C, displaying physiological acclimations to PO₄^3? limitation. Concerning maximal APA per cell, the observation of an all‐or‐nothing pattern along the dilution gradient suggests that synthesis of AP was induced and maximized at the cellular scale as soon as PO₄^3? limitation set in. APA variations revealed that the synthesis of AP was repressed over a PO₄^3? threshold between 0.4 and 1 μM. As lower PO₄^3? concentrations are regularly observed during A. catenella blooms in Thau lagoon, a significant portion of P uptake by A. catenella cells in the field may come from organic compounds.     

UPTAKE OF NITRATE,AMMONIUM, AND UREA BY NITROGEN-STARVED CULTURES OF MICROMONAS PUSILLA (PRASINOPHYCEAE): TRANSIENT RESPONSES1

Nitrogen uptake rates were measured as a function of time following saturating additions (15 μMg-at N·^?1) of ¹⁵N-labelid ammonium, urea, and nitrate to N-starved cultures of the picoflagellate Micromonas pusilla Butcher. Uptake rates were estimated from both the accumulation of ¹⁵N into the cells and the disappearance of nitrogen from the medium. Transient elevated (surge) uptake rates of NH₄⁺ and urea were observed after enrichment. During the first 5 min the initial urea and NH₄⁺ uptake rates were 2- and 4-fold greater than the maximum growth rate (μM_max)observed prior to No₃^? depletion in the cultures. The elevated urea uptake rates declined quickly to a relatively constant value, whereas the initial rates of NH₄⁺ uptake declined rapidly but were followed by a subsequent increase prior to remaining roughly constant. Nitrate was not taken up as readily by N-starved M. pusilla as the reduced N forms. Although NO₃⁺ uptake commenced immediately after enrichment (i.e. no lag period) the N-Specific rate over the next 6 h averaged half the μM_max observed during NO₃^? replete conditions.     

NITROGEN UPTAKE AND ASSIMILATION KINETICS IN ALEXANDRIUM MINUTUM (DYNOPHYCEAE): EFFECT OF N‐LIMITED GROWTH RATE ON NITRATE AND AMMONIUM INTERACTIONS1

Uptake and assimilation kinetics of nitrate and ammonium were investigated along with inhibition of nitrate uptake by ammonium in the harmful dinoflagellate Alexandrium minutum Halim at different nitrogen (N)–limited growth rates. Alexandrium minutum had a strong affinity for nitrate and ammonium (K_s=0.26±0.03 and 0.31±0.04 μmol·L^?1, respectively) whatever the degree of N deficiency of the cells. Ammonium was always the preferred form of nitrogen taken up (=0.42–0.50). In the presence of both forms, nitrate uptake was inhibited by ammonium, and inhibition was particularly marked in N‐sufficient cells (I_max～0.9 and K_i=0.31–0.56 μmol·L^?1). In the case of N assimilation, ammonium was also the preferred form in N‐deficient cells (=0.54–0.72), whereas in N‐sufficient cells, both N sources were equally preferred (=0.90–1.00). The comparison of uptake and assimilation rates highlighted the ability of A. minutum to significantly store in 1 h nitrate and ammonium in amounts sufficient to supply twice the daily N requirements of the slowest‐growing N‐deficient cells. Nitrogen uptake kinetic parameters of A. minutum and their ecological implications are discussed.     

KINETICS OF NITRATE,AMMONIUM, AND UREA UPTAKE BY FOUR INTERTIDAL SEAWEEDS FROM NEW ZEALAND1

The competitive ability for N uptake by four intertidal seaweeds, Stictosiphonia arbuscula (Harvey) King et Puttock, Apophlaea lyallii Hook. f. et Harvey, Scytothamnus australis Hook. f. et Harvey, and Xiphophora gladiata (Labillardière) Montagne ex Harvey, from New Zealand is described by the uptake kinetics for NO₃^?, NH₄⁺, and urea. This is the first study to report uptake kinetics for N uptake by a range of southern hemisphere intertidal seaweeds in relation to season and zonation. Species growing at the highest shore positions had higher NO₃^? and urea uptake at both high and low concentrations and had unsaturable NH₄⁺ uptake in both summer and winter. Although there was evidence of some feedback inhibition of V_max for NO₃^? uptake by Stictosiphonia arbuscula growing at the lower vertical limits of its range, rates were high compared with species growing lower on the shore. Our results highlight the superior competitive ability for N uptake of certain high intertidal seaweeds, and consistent with our previous findings we can conclude that intertidal seaweeds in southeast New Zealand are adapted to maximizing N acquisition in a potentially N‐limiting environment.     

METABOLIC REGULATION OF AMMONIUM UPTAKE BY ULVA RIGIDA (CHLOROPHYTA): A COMPARTMENTAL ANALYSIS OF THE RATE-LIMITING STEP FOR UPTAKE1

Non-linear time courses of ammonium (NH₄⁺) depletion from the medium and internal accumulation of soluble nitrogen (N) in macroalgae imply that the rate-limiting step for ammonium uptake changes over time. We tested this hypothesis by measuring the time course of N accumulation in N-limited Ulva rigida C. Agardh. Total uptake was measured as removal of NH₄⁺ from medium. Rates for the component processes (transport of NH₄⁺ across the membrane = R_v assimilation of tissure NH₄⁺ into soluble N compounds = R_a, assimilation of tissue NH₄⁺ into soluble N compounds = R_a and incorporation of soluble N compounds into macromolecules = R₁) were determined by measuring the rate of labelling of the major tissue N pools after the addition of ¹⁵N-ammonium. The results indicate that nitrogen-specific rates (mass N taken up / mass N present / unit time) are ranked in the order of R_t < R_a < R₁ Absolute uptake rates (μmol N. mg dry wt^?1. h^?1) showed a different relationship. Membrane transport appears to be inhibited when NH₄⁺ accumulates in the tissue. Maximum uptake rates occur when assimilation of NH₄⁺ into soluble N compounds begins. Assimilation of NH₄⁺ into soluble N compounds was initially faster than incorporation of soluble N compounds into macromolecules. Implications of rate limitations caused by differences in maximal rates and maximal pool sizes are discussed.     

UPTAKE OF METHYLAMINE (AN AMMONIUM ANALOGUE) BY MACROCYSTIS PYRIFERA (PHAEOPHYTA)1

The ammonium analogue, methylamine, is taken up rapidly from dilute solution by Macrocystis pyrifera (L.) C. A. Agardh. ¹⁴C-methylamine was used to characterize the transport system, with respect to dependence on external concentration, temperature, pH and substrate specificity. The results suggest that methylamine enters the algal tissue via a specific mediated transport system. Uptake of methylamine showed no consistent relation to the N content of the plant tissue, but was highly dependent on the portion of plant sampled and severely affected by cutting the tissue. The strong inhibition of methylamine uptake by ammonium and lesser inhibition by other alkylamines suggests that the uptake system functions as an “ammonium permease”. Uptake of ¹⁴C-methylamine can be used as a highly sensitive measure of NH₄⁺ uptake activity and should be a useful tool for studying NH₄⁺ uptake in the laboratory and field.     

INHIBITORY EFFECTS OF CONCANAVALIN A AND TUNICAMYCIN ON SEXUAL ATTACHMENT OF ALEXANDRIUM CATENELLA (DINOPHYCEAE)1

The effect of concanavalin A (a lectin) and tunicamycin (an inhibitor of protein glycosylation) on sexual attachment of gamete pairs of the dinoflagellate Alexandrium catenella Whedon & Kofoid was studied. Concanavalin A inhibited sexual attachment at a concentration of 0.005%, and this inhibition was released by the addition of glucose or mannose at 10 mM. Mating type plus cells of A. catenella treated with tunicamycin for 12 h were not capable of sexual attachment. These results are the first to suggest the existence of a cell–cell recognition system in sexual attachment in A. catenella.     

SHORT TERM UPTAKE OF NUTRIENTS BY ENTEROMORPHA PROLIFERA (CHLOROPHYCEAE)1

Rates of NH₄⁺ and NO₃^? uptake were determined by accumulation of ¹⁵N in plant tissue and by disappearance of nutrient from the medium. Agreement between rates calculated by the two methods was good, averaging 82.7% (SD = 15.8%) and 91.2% (SD = 13.7%) for NH₄⁺ and NO₃^? uptake, respectively. An average of 93.4 and 96.0% of added ¹⁵NH₄⁺ and ¹⁵NO₃^? was recovered from the medium and /or plant tissue at the end of the incubations. Both bacterial uptake and regeneration of NH₄⁺ may contribute to discrepancies between NH₄⁺ uptake rates calculated by ¹⁵N accumulation and disappearance of NH₄⁺ from the medium. The influence of tissue composition on uptake of NH₄⁺, NO₃^? and PO₄^3- by Enteromorpha prolifera (Müller) J. Agardh was examined. For NH₄⁺ uptake, V_max was 188 μmol NH₄^+. g dry wt^?1. h^?1 and K_s ranged from 9.3 to 13.4 μM, but there was no correlation between kinetic parameters and tissue nitrogen content. For NO₃^?, both kinetic parameters were higher for plants with low tissue nitrogen than for plants with high tissue nitrogen. Maximum rates were 169 and 75.4 μmol NO₃^?. g dry wt^?1. h^?1, and K_s was 13.3 and 2.31 μM for low and high tissue nitrogen plants, respectively. Estimates of uptake in the field suggested that NH₄⁺ accounted for 65% and NO₃^? for up to 35% of total nitrogen uptake during the summer. Nutrient uptake rates of field-collected plants also indicated that E. prolifera in Yaquina Bay, Oregon was not likely to have been nitrogen-limited, but may have been phosphorus-limited.     

METABOLIC AND ECOLOGICAL CONSTRAINTS IMPOSED BY SIMILAR RATES OF AMMONIUM AND NITRATE UPTAKE PER UNIT SURFACE AREA AT LOW SUBSTRATE CONCENTRATIONS IN MARINE PHYTOPLANKTON AND MACROALGAE1

Marine phytoplankton and macroalgae acquire important resources, such as inorganic nitrogen, from the surrounding seawater by uptake across their entire surface area. Rates of ammonium and nitrate uptake per unit surface area were remarkably similar for both marine phytoplankton and macroalgae at low external concentrations. At an external concentration of 1 μM, the mean rate of nitrogen uptake was 10±2 nmol·cm^?2·h^?1 (n=36). There was a strong negative relationship between log surface area:volume (SA:V) quotient and log nitrogen content per cm² of surface (slope=?0.77), but a positive relationship between log SA:V and log maximum specific growth rate (μ_max; slope=0.46). There was a strong negative relationship between log SA:V and log measured rate of ammonium assimilation per cm² of surface, but the slope (?0.49) was steeper than that required to sustain μ_max (?0.31). Calculated rates of ammonium assimilation required to sustain growth rates measured in natural populations were similar for both marine phytoplankton and macroalgae with an overall mean of 6.2±1.4 nmol·cm^?2·h^?1 (n=15). These values were similar to maximum rates of ammonium assimilation in phytoplankton with high SA:V, but the values for algae with low SA:V were substantially less than the maximum rate of ammonium assimilation. This suggests that the growth rates of both marine phytoplankton and macroalgae in nature are often constrained by rates of uptake and assimilation of nutrients per cm² surface area.     

KINETICS OF NITROGEN-15 LABELLED AMMONIUM UPTAKE BY CAULERPA CUPRESSOIDES (CHLOROPHYTA)1,2,3

The incorporation of ¹⁵NH₄⁺ as a function of time and concentration was used to estimate ammonium uptake by Caulerpa cupressoides (West) C. Agardh, taken from its habitat on the sediments of Tague Bay lagoon, St. Croix, U.S. Virgin Islands. ¹⁵N-based uptake rates followed Michaelis-Menten type saturation kinetics; the maximum uptake rate was 8.7 ± 3.0 μmol N/g dry wt·h at 26° C and the half-saturation constant was 48 ± 10 μM (X?± SE). The high half-saturatiaon constant reflects the dependence of Caulerpa on sediment pore waters as a nitrogen source. Calculations of uptake from isotope time course data were compared to estimates made from ammonium depletion. More ammonium disappeared than could be accounted for by the incorporation of ¹⁵N in Caulerpa, and isotope dilution of the ammonium pool is shown not to be responsible for underestimates of uptake, primarily because large ¹⁵N additions (40–300 μM) were used. It is suggested that either (1) a secondary ammonium sink such as wall sorption or bacterial uptake significantly influenced ammonium concentrations, or (2) ¹⁵N was lost as labelled dissolved organic nitrogen or volatilized during ¹⁵N sample preparation.     

FLUORESCENCE IN SITU HYBRIDIZATION USING rRNA‐TARGETED PROBES FOR SIMPLE AND RAPID IDENTIFICATION OF THE TOXIC DINOFLAGELLATES ALEXANDRIUM TAMARENSE AND ALEXANDRIUM CATENELLA1

The toxic marine dinoflagellates Alexandrium tamarense (Lebor) Balech and A. catenella (Whedon and Kofoid) Taylor have been mainly responsible for paralytic shellfish poisoning in Japan. Rapid and precise identification of these algae has been difficult because this genus contains many morphologically similar toxic and nontoxic species. Here, we report a rapid, precise, and quantitative identification method using three fluorescent, rRNA‐targeted, oligonucleotide probes for A. tamarense (Atm1), A. catenella (Act1), and the nontoxic A. affine (Inoue et Fukuyo; Aaf1). Each probe was species specific when applied using fluorescence in situ hybridization (FISH). None of the probes reacted with three other Alexandrium spp., A. lusitanicum Balech, A. ostenfeldii (Paulsen) Balech & Tangen, and A. insuetum Balech, or with eight other microalgae, including Gymnodinium mikimotoi Miyake et Kominami ex Oda and Heterosigma akashiwo (Hada) Hara et Chihara, suggesting that the species specificity of each probe was very high. Cells labeled with fluorescein 5‐isothiocyanate–conjugated probes showed strong green fluorescence throughout the whole cell except for the nucleus. FISH could be completed within 1 h and largely eliminated the need for identifying species based on key morphological criteria. More than 80% of targeted cells of both species could be identified by microscopy and quantified during growth up to the early stationary phase; more than 70% of cells could be detected in the late stationary phase. The established FISH protocol was found to be a specific, rapid, precise, and quantitative method that might prove to be a useful tool to distinguish and quantify Alexandrium cells collected from Japanese coastal waters.     

CONTRASTING EFFECTS OF METHIONINE SULFOXIMINE ON UPTAKE AND ASSIMILATION OF AMMONIUM IN ULVA INTESTINALIS (CHLOROPHYCEAE)1

Ammonium is assimilated in algae by the glutamine synthetase (GS)–glutamine:2‐oxoglutarate aminotransferase pathway. In addition to the assimilation of external ammonium taken up across the cell membrane, an alga may have to reassimilate ammonium derived from endogenous sources (i.e. nitrate reduction, photorespiration, and amino acid degradation). Methionine sulfoximine (MSX), an irreversible inhibitor of GS, completely inhibited GS activity in Ulva intestinalis L. after 12 h. However, assimilation of externally derived ammonium was completely inhibited after only 1–2 h in the presence of MSX and was followed by production of endogenous ammonium. However, endogenous ammonium production in U. intestinalis represented only a mean of 4% of total assimilation attributable to GS. The internally controlled rate of ammonium uptake (V_i) was almost completely inhibited in the presence of MSX, suggesting that V_i is a measure of the maximum rate of ammonium assimilation. After complete inhibition of ammonium assimilation in the presence of MSX, the initial or surge (V_s) rate of ammonium uptake in the presence of 400 μM ammonium chloride decreased by only 17%. However, the amount that the rate of ammonium uptake decreased by was very similar to the uninhibited rate of ammonium assimilation. In addition, the decrease in the rate of ammonium uptake in darkness (in the absence of MSX) in the presence of 400 μM ammonium chloride matched the decrease in the rate of ammonium assimilation. However, in the presence of 10 μM ammonium chloride, MSX completely inhibited ammonium assimilation but had no effect on the rate of uptake.     

NUTRITIONAL STUDIES OF TWO RED ALGAE. II. KINETICS OF AMMONIUM AND NITRATE UPTAKE1, 2

Similar NH₄₊ and NO₃^?.uptake kinetic patterns were observed in Neoagardhiella baileyi (Harvey ex Kiitzing) Wyinne & Taylor and Gracilaria foliifera (Forssk?l) Borgesen. NO₃^? was taken up in a rate-sturating fashion described by the Michaelis-Menten equation. NH₄₊ uptake was multicomponent: a saturable component was accompanied by a diffusive or a high K component showing no evidence of saturation (at ≤50 μM [NH₄₊]). Nitrogen starved plantsi(C/N atom ratios > ca. 10) showed higher transient rates of NH₄₊ uptake at a given concentration than plants not N-Iimited. Only plants with high N content exhibited diel changes inNH₄₊ uptake rates, and showed transient rates of NH₄₊ accumulation which did not greatly exceed the capacity to incorporate N in steady-state growth. NH₄₊ was preferred over NO_3?even in plants preconditioned on NO_3?as the sole N. source, NO_3? uptake was suppressed at 5μM [NH₄₊], but simultaneous uptake occurred at unsurpressed rates at lower concentrations. Potential for N accumulation was greater via NH₄₊uptake than via NO_3?uptake. Changing capacity for NH₄₊ uptake with N content appears to be a mechanism whereby excessive accumulation of N was avoided by N-.satiated plants but a large accumulation was possible for N-depleted plants.     

AMINO ACID AND UREA UPTAKE IN TEN SPECIES OF CHLOROPHYTA1

The Uptake of radioartively-labelled mixed amino acids, arginine, lysine, leucine, glutamic acid and urea was examined in six species of Volvocales and four species of Chlarococcales grown in nitrate-containing medium. Nonradioactive amino acids in excess were used to estimate specificity of amino and carriers in selected cases. All ten species possess salurable (hence, carrier-mediated) systems for uptake of both arginine and urea. In all Volvacales and one Chlorococcales, the arginine-speciftc carrier (which also transported lysine with lower efficiency) was the only amino acid carrier detected. Three species of Chlororoccales appear to possess a separate carrier for lysine and two of these appear to possess at least one additional carrier that is involved in uptake of non-basic amino acids.     

PHASED OSCILLATIONS IN CELL NUMBERS AND NITRATE IN BATCH CULTURES OF ALEXANDRIUM TAMARENSE (DINOPHYCEAE)1

Alexandrium tamarense (M. Lebour) Balech strains isolated in spring 2007 from a single bloom in Thau lagoon have been grown in nonaxenic artificial media. For three strains showing large oscillations in biomass (crashes followed by recoveries) on a scale of several days, a significant relationship was observed between changes in cell densities (as in vivo fluorescence) and changes in nitrate concentrations. Increases in cell densities were accompanied by decreases in nitrate, while decreases in cell densities corresponded to increases in nitrate, presumably due to nitrification. Net increases in nitrate could reach up to 15 μmol N · L^?1 · d^?1 indicating a very active nitrifying archaeal/bacterial population. However, following population crashes, algal cells can recover and attain biomass levels similar to those reached during the first growth phase. This finding indicates that those archaea/bacteria do not compete for nutrients or do not hamper algal growth under those conditions. In contrast to diatoms, dinoflagellates such as A. tamarense do not excrete/exude dissolved organic matter, thus preventing excessive bacterial growth. This mechanism could help explain the recovery of this species in the presence of bacteria.     

VARIABILITY IN NITRATE UPTAKE KINETICS IN THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE)1

Short-term (1–9 min) nitrate uptake kinetics were measured in Thalassiosira pseudonana (Hust.) Hasle & Heimdal grown in nitrate-limited, ammonium-limited, and nitrate-sufficient continuous cultures. For all cultures, maximal nitrate uptake rates did not develop until approximately 3 min after nitrate addition; thereafter, nitrate uptake rates remained constant or declined slightly. The K_s and V_max for the nitrate-limited cultures were higher at any growth rate than those for the ammonium-limited or nitrate-sufficient cultures. Thus, much higher nitrate concentrations would be required to saturate nitrate uptake in nitrate-limited Thalassiosira pseudonana than is usually considered necessary. The lack of data for other species grown under a range of environmental conditions makes it difficult to generalize about the effect of preconditioning on nitrate uptake kinetics.     

AMMONIUN AND NITRATE UPTAKE BY THE MARINE MACROPHYTES HYPNEA MUSVUFORMIS (RHODOPHYTA) AND MACROCYSTIS PYRIFERA (PHAEOPHYTA)1, 2

NH₄⁺ and NO₃^? uptake were measured by continuous sampling with an autoanalyzer. For Hypnea musciformis (Wulfen) Lamouroux, NO₃^?up take followed saturable kinetics (K₂=4.9 μg-at N t^?1, V_max= 2.85 μg- at N, g(wet)^?1. h^?1. The ammonium uptake data fit a trucatd hyperbola, i.e., saturation was not reach at the concentrations used. NO₃^? uptake was reduced one-half in the presence of NH₄⁺, but presence of NO₃^? had no effect on NH₄⁺ uptake. Darkness reduced both NO₃^? and NH₄⁺ uptake by one-third to one-half. For Macrocystis pyrufera (L) C. Agardh, NO₃^? uptake followed saturable kinetices: K₂=13.1 μg-at N. l^?1. V_max=3.05 μg-at N. g(wet)^?1. h^?1.NH₄⁺ uptake showed saturable kinetics at concentration below 22 μg-at N l -1 (K₂=5.3 μg-at N.1–1, V_max= 2.38 μg-at N G (wet)^?1.h^?1: at higher concentration uptake increased lincarly with concentrations. NO₃^?and NH₄⁺ were taken up simulataneously: presence of one form did not affect uptake of the other.     

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.