REPLY TO COMMENT ON THE LIFE CYCLE AND TOXICITY OF PFIESTERIA PISCICIDA REVISITED1

Free‐living, marine dinoflagellates are typified by a well‐defined, haplontic life cycle with relatively few stages. The most unusual departure from this life cycle is one reported for the heterotrophic dinoflagellate Pfiesteria piscicida Steidinger et Burkholder. This species is alleged to have at least 24 life cycle stages including amoebae and a chrysophyte‐like cyst form ( Burkholder et al. 1992 , Burkholder and Glasgow 1997a ) not previously known in free‐living marine dinoflagellates. Litaker et al. (2002) redescribed the life cycle of P. piscicida from single‐cell isolates and found only life cycle stages typical of free‐living marine dinoflagellates. The discrepancy between these observations and the life cycle reported in the literature prompted a rigorous study to resolve the life cycle of P. piscicida. Burkholder and Glasgow (2002) took exception to this study, arguing that Litaker et al. (2002) misunderstood the life cycle of P. piscicida and ignored recent publications. We present a rebuttal of their criticisms and suggest a simple way to resolve the discrepancies in the P. piscicida life cycle.     

Trophic Controls on Stage Transformations of a Toxic Ambush-Predator Dinoflagellate1

ABSTRACT. The toxic dinoflagellate, Pfiesteria piscicida, was recently implicated as the causative agent for about 50% of the major fish kills occurring over a three-year period in the Albemarle-Pamlico Estuarine System of the southeastern USA. Transformations between life-history stages of this dinoflagellate are controlled by the availability of fresh fish secretions or fish tissues, and secondarily influenced by the availability of alternate prey including bacteria, algae, microfauna, and mammalian tissues. Toxic zoospores of P. piscicida subdue fish by excreting lethal neurotoxins that narcotize the prey, disrupt its osmoregulatory system, and attack its nervous system. While prey are dying, the zoospores feed upon bits of fish tissue and complete the sexual phase of the dinoflagellate life cycle. Other stages in the complex life cycle of P. piscidia include cryptic forms of filose, rhizopodial, and lobose amoebae that can form within minutes from toxic zoospores, gametes, or planozygotes. These cryptic amoebae feed upon fish carcasses and other prey and, thus far, have proven less vulnerable to microbial predators than flagellated life-history stages. Lobose amoebae that develop from toxic zoospores and planozygotes during colder periods have also shown ambush behavior toward live fish. In the presence of abundant flagellated algal prey, amoeboid stages produce nontoxic zoospores that can become toxic and form gametes when they detect what is presumed to be a threshold level of a stimulatory substance(s) derived from live fish. The diverse amoeboid stages of this fish “ambush-predator” and at least one other Pfiesteria-like species are ubiquitous and abundant in brackish waters along the western Atlantic and Gulf Coasts, indicating a need to re-evaluate the role of dinoflagellates in the microbial food webs of turbid nutrient-enriched estuaries.     

THE LIFE CYCLE AND TOXICITY OF PFIESTERIA PISCICIDA REVISITED1

Despite use of excellent molecular techniques, Litaker et al. (2002) cannot provide insights about the life history of toxic Pfiesteria piscicida because they showed no data in support of having used toxic strains; rather they presented evidence that they used non‐inducible strains. Litaker et al. did not find amoeboid stages or a chrysophyte‐like cyst stage in several cultures and unequivocally concluded that the stages do not exist in all P. piscicida strains. Thus, they did not consider the tenet that absence of evidence does not constitute proof of absence. Apparent discrepancies between the research by Litaker et al. and previous research on Pfiesteria can be resolved as follows: First, Litaker et al. did not use toxic strains. We have reported findings (similar to Litaker et al.) showing few amoeboid transformations in non‐inducible strains, which manifest some but not all of the forms that have been documented in some toxic strains. We, and others, have documented active toxicity to fish, transformations to amoebae, and chrysophyte‐like cysts in some clonal toxic strains. Second, the data from several recent publications, which were available but not mentioned by Litaker et al. or by Coats (2002) in accompanying commentary, have verified P. piscicida amoebae, chrysophyte‐like cysts, and other stages in some toxic strains through a combination of approaches including PCR data from clonal cultures.     

Predator/Prey Interaction between <Emphasis Type="Italic">Pfiesteria piscicida</Emphasis> and <Emphasis Type="Italic">Rhodomonas</Emphasis> Mediated by a Marine Alpha Proteobacterium

The dinoflagellate Pfiesteria piscicida coexists with bacteria in aquatic environments and as such, may interact with them at the physiological level. This study was designed to investigate the influence of bacteria, present in a clonal culture of Pfiesteria piscicida, on the predator/prey relationship of this dinoflagellate with the alga Rhodomonas. A series of replenishment experiments with bacteria isolated from P. piscicida clonal culture and the bacteria-free P. piscicida derived from the same culture were carried out. In the presence of bacteria, the number of P. piscicida increased significantly when incubated with alga Rhodomonas. This enhanced growth was almost entirely due to the increased consumption rate of Rhodomonas by P. piscicida since in bacteria-free (axenic) cultures Rhodomonas were consumed at significantly reduced rates relative to cultures with bacteria. Subsequent replenishment experiments with individual bacterial isolates showed that a single isolate was responsible for the increased predation rate of P. piscicida. The presence or absence of this specific bacterium determined the outcome of the interaction between P. piscicida and Rhodomonas. Partial sequence analysis of the 16S rDNA of this isolate indicated that it was a novel marine alpha proteobacterium with sequence similarities to a Roseobacter sp. and a bacterium recently isolated from a toxic dinoflagellate Alexandrium sp.     

THE SEXUAL LIFE CYCLES OF PFIESTERIA PISCICIDA AND CRYPTOPERIDINIOPSOIDS (DINOPHYCEAE)1

Sexual life cycle events in Pfiesteria piscicida and cryptoperidiniopsoid heterotrophic dinoflagellates were determined by following the development of isolated gamete pairs in single‐drop microcultures with cryptophyte prey. Under these conditions, the observed sequence of zygote formation, development, and postzygotic divisions was similar in these dinoflagellates. Fusion of motile gamete pairs each produced a rapidly swimming uninucleate planozygote with two longitudinal flagella. Planozygotes enlarged as they fed repeatedly on cryptophytes. In <12 h in most cases, each planozygote formed a transparent‐walled nonmotile cell (cyst) with a single nucleus. Zygotic cysts did not exhibit dormancy under these conditions. In each taxon, dramatic swirling chromosome movements (nuclear cyclosis) were found in zygote nuclei before division. In P. piscicida, nuclear cyclosis occurred in the zygotic cyst or apparently earlier in the planozygote. In the cryptoperidiniopsoids, nuclear cyclosis occurred inthe zygotic cyst. After nuclear cyclosis, a single cell division occurred in P. piscicida and cryptoperidiniopsoid zygotic cysts, producing two offspring that emerged as biflagellated cells. These two flagellated cells typically swam for hours and fed on cryptophytes before encysting. A single cell division in these cysts produced two biflagellated offspring that also fed before encysting for further reproduction. This sequence of zygote development and postzygotic divisions typically was completed within 24 h and was confirmed in examples from different isolates of each taxon. Some aspects of the P. piscicida sexual life cycle determined here differed from previous reports.     

A NEW LARVAL FISH BIOASSAY FOR TESTING THE PATHOGENICITY OF PFIESTERIA SPP. (DINOPHYCEAE)1

Water quality, microbial contamination, prior fish health, and variable results have been major impediments to identifying the cause and mechanism of fish mortality in standard aquarium‐format Pfiesteria bioassays. Therefore, we developed a sensitive 96‐h larval fish bioassay for assessing Pfiesteria spp. pathogenicity using six‐well tissue culture plates and 7‐day‐old larval cyprinodontid fish. We used the assay to test pathogenicity of several clonal lines of Pfiesteria piscicida Steidinger and Burkholder and P. shumwayae Glasgow and Burkholder that had been cultured with algal prey for 2 to 36 months. The P. shumwayae cultures exhibited 80%–100% cumulative mortality in less than 96 h at initial zoospore densities of approximately 1000 cells·mL^?1. No fish mortalities occurred with P. piscicida at identical densities or in controls. In a dose‐response assay, we demonstrated a strong positive correlation between dinospore density and fish mortality in a highly pathogenic culture of P. shumwayae, generating a 96‐h LD₅₀ of 108 zoospores·mL^?1. Additionally, we applied the assay to evaluate a 38‐L P. shumwayae bioassay that was actively killing fish and compared results with those from exposures of juvenile tilapia (Oreochromis niloticus) in a 500‐mL assay system. Water from the fish‐killing 38‐L assay was filtered and centrifuged to produce fractions dominated by dinoflagellates, bacteria, or presumed ichthyotoxin (cell‐free fraction). After 96 h, the larval fish assay exhibited 50%–100% cumulative mortality only in fractions containing dinoflagellates, with no mortalities occurring in the other fractions. The 500‐mL bioassay with tilapia produced inconsistent results and demonstrated no clear correlation between mortality and treatment. The new larval fish bioassay was demonstrated as a highly effective method to verify and evaluate dinoflagellate pathogenicity.     

Detection of a novel ecotype of <Emphasis Type="Italic">Pfiesteria piscicida</Emphasis> (Dinophyceae) in an Antarctic saline lake by real-time PCR

The heterotrophic dinoflagellate Pfiesteria piscicida was detected in Ace Lake in the Vestfold Hills, eastern Antarctica by using real-time PCR based on 18S rDNA sequences. Antarctic water samples collected in 2004 were tested by species-specific real-time PCR assays for the identification of P. piscicida and P. shumwayae. Positive results were shown with P. piscicida-specific real-time PCR, and PCR products were examined by sequence analysis for confirmation. A phylogenetic tree made from partial 18S rDNA sequences showed that the Antarctic clone clustered with P. piscicida. This result suggests that P. piscicida is present in the extreme conditions of an Antarctic saline lake which has not contained fish for thousands of years.     

Karyology of a Marine Non-Motile Dinoflagellate, Pyrocystis lunula

Dinoflagellates have a unique and interesting intracellular architecture such as permanently condensed chromosomes throughout the cell cycle. However the study of dinoflagellate chromosomes is not amendable because of the unusually higher number of chromosomes and problems in sample preparation. The species of Pyrocystis spend most of their life cycle as vegetative cyst forms and have been used as experimental organisms for bioluminescence and circadian rhythms. Here, we documented the content of DNA in different life stages and the chromosome karyology in a marine non-motile dinoflagellate Pyrocystis lunula, through light and fluorescent microscopy, serial ultra-thin sectioning, and three dimension (3D) modeling. The DNA content doubles during DNA synthesis and in the end of the cell division two separate daughter cells have the approximately same fluorescent values for the mother cells. Using serial ultra-thin sectioning and 3D modeling, we report the first ultrastructural karyogram. The cells chosen were at the end of karyokinesis. A total of 98 chromosomes were counted and assigned to 49 pairs. In this species, DNA synthesis appears to occur before, or during asexual division and P. lunula lives a diplontic life cycle.     

Differences in pigmentation between life cycle stages in Scrippsiella lachrymosa (dinophyceae)

Various life cycle stages of cyst‐producing dinoflagellates often appear differently colored under the microscope; gametes appear paler while zygotes are darker in comparison to vegetative cells. To compare physiological and photochemical competency, the pigment composition of discrete life cycle stages was determined for the common resting cyst‐producing dinoflagellate Scrippsiella lachrymosa. Vegetative cells had the highest cellular pigment content (25.2 ± 0.5 pg · cell^?1), whereas gamete pigment content was 22% lower. The pigment content of zygotes was 82% lower than vegetative cells, even though they appeared darker under the microscope. Zygotes of S. lachrymosa contained significantly higher cellular concentrations of β‐carotene (0.65 ± 0.15 pg · cell^?1) than all other life stages. Photoprotective pigments and the de‐epoxidation ratio of xanthophylls‐cycle pigments in S. lachrymosa were significantly elevated in zygotes and cysts compared to other stages. This suggests a role for accessory pigments in combating intracellular oxidative stress during sexual reproduction or encystment. Resting cysts contained some pigments even though chloroplasts were not visible, suggesting that the brightly colored accumulation body contained photosynthetic pigments. The differences in pigmentation between life stages have implications for interpretation of pigment data from field samples when sampled during dinoflagellate blooms.     

EVIDENCE FOR ASEXUAL RESTING CYSTS IN THE LIFE CYCLE OF THE MARINE PERIDINOID DINOFLAGELLATE,SCRIPPSIELLA HANGOEI1

Scrippsiella hangoei (Schiller) Larsen is a peridinoid dinoflagellate that grows during winter and spring in the Baltic Sea. In culture this species formed round, smooth cysts when strains were mixed, indicating heterothallic sexuality and hypnozygote production. However, cysts of the same morphology were also formed in clonal strains exposed to slightly elevated temperature. To better understand the role of cysts in the life cycle of S. hangoei, cyst formation and dormancy were examined in culture experiments and the cellular DNA content of flagellate cells and cysts was compared in clonal and mixed strains using flow cytometry. S. hangoei exhibited a high rate of cyst formation in culture. Cysts produced in both clonal and mixed strain cultures were thick‐walled and underwent a dormancy period of 4 months before germinating. The S. hangoei flagellate cell population DNA distributions consisted of 1C, intermediate, and 2C DNA, indicative of respective eukaryotic cell cycle phases G1, S, and G2M. The majority (>95%) of cysts had a measured DNA content equivalent to the lower 1C DNA value, indicating a haploid nuclear phase and an asexual mode of cyst formation. A small percentage (<5%) of cysts produced in the mixed strain culture had 2C DNA, and thus could have been diploid zygotes. These findings represent the first measurements of dinoflagellate resting cyst DNA content, and provide the first quantitative evidence for dinoflagellate asexual resting cysts. Asexual resting cysts may be a more common feature of dinoflagellate life cycles than previously thought.     

Response of two zooptankton grazers to an ichthyotoxic estuarine dinoflagellate

The dinoflagellate Pfiesteria piscicida (gen. et sp. nov.).a toxic ‘ambush predator’, has been implicated asa causative agent of major fish kills in estuanne ecosystemsof the southeastern USA. Here we report the first experimentaltests of interactions between P.piscicida and estuarine zooplanktonpredators. specifically the rotifer Brachionus plicatilis andthe calanoid copepod Acartia tonsa. Short-term (10 day) exposureof adult B.plicatilis to P.piscicida as a food resource, aloneor in combination with the non-toxic green algae Nannochlorisand Tetraselmis. did not increase rotifer mortality relativeto animals that were given only non-toxic greens Similarly,short-term (3 day) feeding trials using adult A.tonsa indicatedthat the copepods survived equally well on either P.piscicidaor the non-toxic diatom Thalassiosira pseudonana. Copepods giventoxic dinoflagellates exhibited erratic behavior, however, relativeto animals given diatom prey. The fecundity of B.plicatiliswhen fed the toxic dinoflagellate was comparable to or higherthan that of rotifers fed only non-toxic greens We concludethat, on a short-term basis, toxic stages of P.piscicida canbe readily utilized as a nutritional resource by these commonestuarine zooplankters. More long-term effects of P.piscicidaon zooplankton, the potential for toxin bioaccumulation acrosstrophic levels, and the utility of zooplankton as biologicalcontrol agents for this toxic dinoflagellate. remain importantunanswered questions.     

Characterization of the rRNA Locus of Pfiesteria piscicida and Development of Standard and Quantitative PCR-Based Detection Assays Targeted to the Nontranscribed Spacer

Pfiesteria piscicida is a heterotrophic dinoflagellate widely distributed along the middle Atlantic shore of the United States and associated with fish kills in the Neuse River (North Carolina) and the Chesapeake Bay (Maryland and Virginia). We constructed a genomic DNA library from clonally cultured P. piscicida and characterized the nontranscribed spacer (NTS), small subunit, internal transcribed spacer 1 (ITS1), 5.8S region, ITS2, and large subunit of the rRNA gene cluster. Based on the P. piscicida ribosomal DNA sequence, we developed a PCR-based detection assay that targets the NTS. The assay specificity was assessed by testing clonal P. piscicida and Pfiesteria shumwayae, 35 additional dinoflagellate species, and algal prey (Rhodomonas sp.). Only P. piscicida and nine presumptive P. piscicida isolates tested positive. All PCR-positive products yielded identical sequences for P. piscicida, suggesting that the PCR-based assay is species specific. The assay can detect a single P. piscicida zoospore in 1 ml of water, 10 resting cysts in 1 g of sediment, or 10 fg of P. piscicida DNA in 1 μg of heterologous DNA. An internal standard for the PCR assay was constructed to identify potential false-negative results in testing of environmental sediment and water samples and as a competitor for the development of a quantitative competitive PCR assay format. The specificities of both qualitative and quantitative PCR assay formats were validated with >200 environmental samples, and the assays provide simple, rapid, and accurate methods for the assessment of P. piscicida in water and sediments.     

Feeding preferences and grazing rates of Pfiesteria piscicida and a cryptoperidiniopsoid preying on fish blood cells and algal prey

The grazing rates and feeding preferences of the dinoflagellates Pfiesteria piscicida and a cryptoperidiniopsoid on the alga Rhodomonas sp. and fish blood cells were calculated at different ratios of the two food types and at different total food densities. Data from 6 h grazing periods within microcosms were used to calculate grazing rates. Grazing rates of both dinoflagellates increased linearly with an increased ratio of blood cells to Rhodomonas, and P. piscicida had a higher maximum grazing rate than the cryptoperidiniopsoid. The grazing rate of P. piscicida on Rhodomonas also increased with increased Rhodomonas densities relative to the blood cells, but increased densities of Rhodomonas did not increase the grazing rate of the cryptoperidiniopsoid, suggesting a lower feeding threshold for this species. Both dinoflagellates demonstrated a preference for fish blood cells over Rhodomonas cells, with no significant difference in the index of preference between the two species. Total food abundance affected the degree of preference differently for each dinoflagellate species. A higher index of feeding preference was attained by P. piscicida when resource levels were high, while the cryptoperidiniopsoid did not show this response. A preference for fish blood cells occurred at all food ratios for both dinoflagellates, including when blood cells were scarce relative to the alternate food type (15% of total available food). These results suggest that these strains of P. piscicida and the cryptoperidiniopsoid share similar feeding preferences for the prey types tested, although cryptoperidiniopsoids have not been associated with fish kills.     

Distribution of Pfiesteria piscicida cyst populations in sediments of the Delaware Inland Bays, USA

The toxic dinoflagellate, Pfiesteria piscicida, is a common constituent of the phytoplankton community in the Delaware Inland Bays, USA. In this study, molecular methods were used to investigate the distributions of benthic stages (cysts) of P. piscicida in sediment cores from the Delaware Inland Bays. Cores from 35 sites were partitioned into nephloid and anoxic layers and analyzed for P. piscicida by nested amplification of the 18S rDNA gene using P. piscicida-specific primers. The presence of inhibitory substances in the PCR reaction was evaluated by inclusion of an exogenous control DNA in the extraction buffer, thus eliminating samples that may yield false-negative results. Our results indicate a patchy distribution of P. piscicida in sediments of the Delaware Inland Bays, with distinct differences between each of the three bays. Overall, P. piscicida was found more frequently in sediments from Rehoboth Bay compared to Indian River and Little Assawoman Bays. These differences suggest (i) that populations of P. piscicida may be more widely distributed in Rehoboth Bay, (ii) that populations of P. piscicida may have been introduced to Rehoboth Bay at an earlier time, (iii) that past blooms of P. piscicida in Rehoboth Bay estuaries may have seeded the sediments with higher numbers of cysts, and/or (iv) that Rehoboth Bay sediments may be more resistant to clearing due to storm turbulence.     

The life cycle of the amoeboid alga Synchroma grande (Synchromophyceae, Heterokontophyta)--highly adapted yet equally equipped for rapid diversification in benthic habitats

Synchroma grande (Synchromophyceae, Heterokontophyta) is a marine amoeboid alga, which was isolated from a benthic habitat. This species has sessile cell stages (amoeboid cells with lorica and cysts) and non‐sessile cell stages (migrating and floating amoebae) during its life cycle. The different cell types and their transitions within the life cycle are described, as are their putative functions. Cell proliferation was observed only in cells attached to the substrate but not in free‐floating or migrating cells. We also characterised the phagotrophy of the meroplasmodium in comparison to other amoeboid algae and the formation of the lorica. The functional adaptations of S. grande during its life cycle were compared to the cell stages of other amoeboid algae of the red and green chloroplast lineages. S. grande was found to be highly adapted to the benthic habitat. One sexual and two asexual reproductive strategies (haplo‐diploid life cycle) support the ability of this species to achieve rapid diversification and high adaptivity in its natural habitat.     

Sterols of the heterotrophic dinoflagellate,pfiesteria piscicida (dinophyceae): is there a lipid biomarker?1

Within U.S. waters, blooms of the dinoflagellate, Pfiesteria piscicida, have been recorded on an almost regular basis in the Chesapeake Bay and surrounding mid‐Atlantic regions for the last two decades. Despite the apparent significance of such blooms to the environment and human health and the attendant economic consequences, little work has addressed the physiology and biochemistry, particularly that of sterol composition, of P. piscicida. GC‐MS characterization of trimethylsilyl ether derivatives of sterols from free sterol and sterol ester fractions was performed in an effort to determine whether P. piscicida produces unique sterols that may serve as potential biomarkers. This characterization revealed that like most dinoflagellates, the majority of sterols was present as free sterols. Furthermore, the profile of free sterols was found to resemble those of photosynthetic dinoflagellates, with the dominant compound being the previously reported dinoflagellate sterol, dinosterol. A number of other 4α‐methyl‐substituted sterols and steroidal ketones common to other dinoflagellates were also identified. No strong candidate(s) for a unique sterol biomarker was present.     

Life Cycle of the pseudocolonial dinoflagellate Polykrikos kofoidii (Gymnodiniales,Dinoflagellata)

The athecate, pseudocolonial polykrikoid dinoflag‐ellates show a greater morphological complexity than many other dinoflagellate cells and contain not only elaborate extrusomes but sulci, cinguli, flagellar pairs, and nuclei in multiple copies. Among polykrikoids, Polykrikos kofoidii is a common species that plays an important role as a grazer of toxic planktonic algae but whose life cycle is poorly known. In this study, the main life cycle stages of P. kofoidii were examined and documented for the first time. The formation of gametes, 2‐zooid‐1‐nucleus stages very different from vegetative cells, was observed and the process of gamete fusion, isogamy, was recorded. Karyogamy followed shortly after completed plasmogamy. A complex reorganization of furrows (cinguli and sulci) and flagella followed zygote formation, resulting in a 4‐zooid zygote with one nucleus. The fate of zygotes under different nutritional conditions was also investigated; well‐fed zygotes were able to reenter the vegetative cycle via meiotic divisions as indicated by nuclear cyclosis. However, nuclear cyclosis was preceded by a presumably mitotic division of the primary zygote nucleus which by definition would imply that P. kofoidii has a diplohaplontic life cycle. Nuclear cyclosis in germlings hatched from spiny resting cysts indicate that these cysts are of zygote origin (hypnozygotes). Hypnozygote formation, cyst hatching, the morphology of the germling (a 1‐zooid cell), and its development into a normal pseudocolony are documented here for the first time. There is evidence that P. kofoidii has a system of complex heterothallism.     

GENETIC VARIATION AMONG STRAINS OF PSEUDOPFIESTERIA SHUMWAYAE AND PFIESTERIA PISCICIDA (DINOPHYCEAE)1

The putatively toxic dinoflagellates Pseudopfiesteria shumwayae (Glasgow et J. M. Burkh.) Litaker, Steid., P. L. Mason, Shields et P. A. Tester and Pfiesteria piscicida Steid. et J. M. Burkh. have been implicated in massive fish kills and of having negative impacts on human health along the mid‐Atlantic seaboard of the USA. Considerable debate still remains as to the mechanisms responsible for fish mortality (toxicity vs. micropredation) caused by these dinoflagellates. Genetic differences among these cultures have not been adequately investigated and may account for or correlate with phenotypic variability among strains within each species. Genetic variation among strains of Ps. shumwayae and P. piscicida was examined by PCR–RFLP analysis using cultures obtained from the Provasoli‐Guillard National Center for Culture of Marine Phytoplankton (CCMP), as well as those from our own and other colleagues’ collection efforts. Examination of restriction digest banding profiles for 22 strains of Ps. shumwayae revealed the presence of 10 polymorphic restriction endonuclease sites within the first and second internal transcribed spacers (ITS1 and ITS2) and the 5.8S gene of the rDNA complex, and the cytochrome oxidase subunit I (COI) gene. Three compound genotypes were represented within the 22 Ps. shumwayae strains. Conversely, PCR–RFLP examination of 14 strains of P. piscicida at the same ITS1, 5.8S, and ITS2 regions revealed only one variable restriction endonuclease site, located in the ITS1 region. In addition, a dinoflagellate culture listed as P. piscicida (CCMP 1928) and analyzed as part of this study was identified as closely related to Luciella masanensis P. L. Mason, H. J. Jeong, Litaker, Reece et Steid.