Growth and epiphytic behavior of three Gambierdiscus species (Dinophyceae) associated with various macroalgal substrates

Species of the benthic dinoflagellate Gambierdiscus produce polyether neurotoxins that caused ciguatera fish/shellfish poisoning in human. The toxins enter marine food webs by foraging of herbivores on the biotic substrates like macroalgae that host the toxic dinoflagellates. Interaction of Gambierdiscus and their macroalgal substrate hosts is believed to shape the tendency of substrate preferences and habitat specialization. This was supported by studies that manifested epiphytic preferences and behaviors in Gambierdiscus species toward different macroalgal hosts. To further examine the supposition, a laboratory-based experimental study was conducted to examine the growth, epiphytic behaviors and host preferences of three Gambierdiscus species towards four macroalgal hosts over a culture period of 40 days. The dinoflagellates Gambierdiscus balechii, G. caribaeus, and a new ribotype, herein designated as Gambierdiscus type 7 were initially identified based on the thecal morphology and molecular characterization. Our results showed that Gambierdiscus species tested in this study exhibited higher growth rates in the presence of macroalgal hosts. Growth responses and attachment behaviors, however, differed among different species and strains of Gambierdiscus over different macroalgal substrate hosts. Cells of Gambierdiscus mostly attached to substrate hosts at the beginning of the experiments but detached at the later time. Localized Gambierdiscus-host interactions, as demonstrated in this study, could help to better inform efforts of sampling and monitoring of this benthic toxic dinoflagellate.     

An examination of the epiphytic nature of Gambierdiscus toxicus, a dinoflagellate involved in ciguatera fish poisoning

Twenty-four specimen of macroalgae were collected in nearshore waters of the island of Hawaii, identified, and maintained to examine how the epiphytic relationship between Gambierdiscus toxicus (isolate BIG12) varied among the macroalgal species. Gambierdiscus cells were introduced to Petri dishes containing 100 g samples of each macroalgal host, which were examined at two, 16, 24, and every 24–72 h thereafter, over a 29-day period. Gambierdiscus proliferated in the presence of some host species (e.g., Galaxaura marginata and Jania sp.), but grew little in the presence of other species (e.g., Portieria hornemannii). Gambierdiscus exhibited high survival rates (>99%) in the presence of Chaetomorpha sp., but died before the end of the experiment (after 21 days) with other host species (e.g., Dictyota and Microdictyon spp.). Gambierdiscus avoided contact with P. hornemannii, but averaged up to 30% attachment with other host species. The numbers of Gambierdiscus cells belonging to one of three classes (alive and attached; alive and unattached; and dead) were determined for each time point. The 24 algal hosts were grouped according to their commonalities relative to these three classes using a Bray-Curtis similarity index, similarity profile (SIMPROF) permutation tests, and Multi-Dimensional Scaling (MDS) analysis (PRIMER 6). The resultant six groupings were used to construct different Gambierdiscus growth profiles for the different algal hosts. Group A is characterized by a preponderance of unattached cells and high mortality rates. Groups B, C, E, and F also displayed high proportions of unattached cells, but mortality either occurred later (Groups B and C) or rates were lower (Groups E and F). Group D had the highest proportion of attached cells. Group E contained three out of the four chlorophyte species, while Group F contained the majority of the rhodophytes. Over 50% of the species in Group F are considered to be palatable, whereas Groups A, B, and C are composed of species that exhibit chemical defenses against herbivory. The results of this study coupled with previous findings indicate that Gambierdiscus is not an obligate epiphyte; it can be free-swimming and found in the plankton. The conditions that lead to changes between epiphytic and planktonic stages need to be better studied in order to determine how they affect Gambierdiscus growth and physiology, connectivity and dispersion mechanisms, and toxin movement up into the foodweb.     

Occurrence of epibenthic dinoflagellates in relation to biotic substrates and to environmental factors in Southern Mediterranean (Bizerte Bay and Lagoon,Tunisia): An emphasis on the harmful Ostreopsis spp., Prorocentrum lima and Coolia monotis

Harmful events associated with epibenthic dinoflagellates, have been reported more frequently over the last decades. Occurrence of potentially toxic benthic dinoflagellates, on the leaves of two magnoliophytes (Cymodocea nodosa and Zostera noltei) and thalli of the macroalgae (Ulva rigida), was monitored over one year (From May 2015 to April 2016) in the Bizerte Bay and Lagoon (North of Tunisia, Southern Mediterranean Sea). The investigated lagoon is known to be highly anthropized. This is the first report on the seasonal distribution of epibenthic dinoflagellates hosted by natural substrates, from two contrasted, adjacent coastal Mediterranean ecosystems. The environmental factors promoting the development of the harmful epibenthic dinoflagellates Ostreopsis spp., Prorocentrum lima and Coolia monotis were investigated. The highest cell densities were reached by Ostreopsis spp. (1.9 × 10³ cells g⁻¹ FW, in October 2015), P. lima (1.6 × 10³ cells g⁻¹ FW, in June 2015) and C. monotis (1.1 × 10³ cells g⁻¹ FW, in May 2015). C. nodosa and Z. noltei were the most favorable host macrophytes for C. monotis (in station L2) and Ostreopsis spp. (in station L3), respectively. Positive correlations were recorded between Ostreopsis spp. and temperature. Densities of the epibenthic dinoflagellates varied according to the collection site, and a great disparity was observed between the Bay and the Lagoon. Maximum concentrations were recorded on C. nodosa leaves from the Bizerte Bay, while low epiphytic cell abundances were associated with macrophytes sampled from the Bizerte Lagoon. The observed differences in dinoflagellate abundances between the two ecosystems (Bay-Lagoon) seemed not related to the nutrients, but rather to the poor environmental conditions in the lagoon.     

A bacterium that inhibits the growth of Pfiesteria piscicida and other dinoflagellates

Toxic dinoflagellate blooms have increased in estuaries of the east coast of the United States in recent years, and the discovery of Pfiesteria piscicida has brought renewed attention to the problem of harmful algal blooms (HAB) in general. Many bacteria and viruses have been isolated that have algicidal or algistatic effects on phytoplankton, including HAB species. Twenty-two bacterial isolates from the Delaware Inland Bays were screened for algicidal activity. One isolate (Shewanella IRI-160) had a growth-inhibiting effect on all three dinoflagellate species tested, including P. piscicida (potentially toxic zoospores), Prorocentrum minimum, and Gyrodinium uncatenum. This bacterium did not have a negative effect on the growth of any of the other four common estuarine non-dinoflagellate species tested, and in fact had a slight stimulatory effect on a diatom, a prasinophyte, a cryptophyte, and a raphidophyte. Shewanella IRI-160 is the first non-microzooplankton example of a microbe with the ability to control and inhibit the growth of P. piscicida, suggesting that bacteria in the natural environment could play a role in controlling the growth and abundance of P. piscicida and other dinoflagellates. Such bacteria could also potentially be used as management tools to prevent the proliferation of potentially harmful dinoflagellates in estuaries and coastal waters.     

THE PAS/ACCUMULATION BODIES IN PROROCENTRUM LIMA AND PROROCENTRUM MACULOSUM (DINOPHYCEAE) ARE DINOFLAGELLATE LYSOSOMES1

Numerous spherical bodies containing electron-dense material, fibrous material, and membranous material are present in the cytoplasm of two dinoflagellate species, Prorocentrum lima (Ehr.) Dodge and Prorocentrum maculosum Faust. Similar bodies have been observed in other dinoflagellates and have been termed accumulation bodies or PAS bodies. In both Prorocentrum species, these bodies autofluoresce under blue light excitation and increase in size with cell culture age. They possess acid phosphatase activity, react positively with the periodic acid/Schiff reagent, and stain with acridine orange. All these properties are characteristic of eukaryotic lysosomes; thus, we propose that dinoflagellate accumulation bodies and PAS bodies are identical organelles and are, in fact, dinoflagellate lysosomes.     

Epiphytic abundance and toxicity of Prorocentrum lima populations in the Fleet Lagoon, UK

Planktonic Dinophysis spp. and epiphytic Prorocentrum lima (Ehrenberg) Dodge are known dinoflagellate producers of okadaic acid (OA) and dinophysistoxins (DTX), causative phycotoxins of diarrhetic shellfish poisoning (DSP). Underestimation of toxic dinoflagellates associated with a toxic event may be due to the lack of sampling of species with epiphytic and epibenthic strategies, such as P. lima. As Dinophysis spp. is not found in the Fleet Lagoon, Dorset, but previous DSP events have closed the Crassostrea gigas oyster farm, P. lima is the most likely causative organism. A field assay for separating microalgal epiphytes and concentrating wild cells on to filters was successfully applied to sub-samples of a variety of macroalgae and macrophytes (seagrass) collected from the Fleet during summer 2002. P. lima was present in increasing cell densities on most substratum species, over the sampling period, from 10² to 10³ cells g⁻¹ fresh weight (FW) plant biomass. LC–MS analysis detected OA and DTX-1 in extracts of wild P. lima cells, in ratios characteristic of P. lima strains previously isolated from the Fleet. No toxins, however, were detected in oyster flesh.     

Dead bleached coral provides new surfaces for dinoflagellates implicated in ciguatera fish poisonings

Synopsis Dead sections of bleached corals in the United States Virgin Islands and British Virgin Islands were found to be colonized by filamentous algae harboring epiphytic dinoflagellates implicated in ciguatera fish poisonings. The dinoflagellates Ostreopsis lenticularis, Prorocentrum concavum, and P. lima were found in association with filamentous algae growing on dead sections of bleached Montastrea annularis and Acropora cervicornis. Several fish species from the families Acanthuridae, Pomacentridae, and Scaridae were observed to readily consume this filamentous algae/epiphytic dinoflagellate food source. Such fishes are common prey for large piscivores inhabiting tropical reefs. An increased incidence of ciguatoxic fishes may occur on reefs where bleaching events have caused significant coral mortality.     

Effects of temperature,salinity and composition of the dinoflagellate assemblage on the growth of Gambierdiscus carpenteri isolated from the Great Barrier Reef

Increases in reported incidence of ciguatera fish poisoning (hereafter ciguatera) have been linked to warmer sea temperatures that are known to trigger coral bleaching events. The drivers that trigger blooms of ciguatera-causing dinoflagellates on the Great Barrier Reef (GBR) are poorly understood. This study investigated the effects of increased temperatures and lowered salinities, often associated with environmental disturbance events, on the population growth of two strains of the potentially ciguatera-causing dinoflagellate, Gambierdiscus carpenteri (NQAIF116 and NQAIF380). Both strains were isolated from the central GBR with NQAIF116 being an inshore strain and NQAIF380 an isolate from a stable environment of a large coral reef aquarium exhibit in ReefHQ, Townsville, Australia. Species of Gambierdiscus are often found as part of a mixed assemblage of benthic toxic dinoflagellates on macroalgal substrates. The effect of assemblage structure of dinoflagellates on the growth of Gambierdiscus populations has, however, not been explored. The study, therefore investigated the growth of G. carpenteri within mixed assemblages of benthic dinoflagellates. Population growth was monitored over a period of 28 days under three salinities (16, 26 and 36) and three temperature (24, 28 and 34 °C) conditions in a fully crossed experimental design. Temperature and salinity had a significant effect on population growth. Strain NQAIF380 exhibited significantly higher growth at 28 °C compared to strain NQAIF116, which had highest growth at 24 °C. When strain NQAIF116 was co-cultured with the benthic dinoflagellates, Prorocentrum lima and Ostreopsis sp., inhibitory effects on population growth were observed at a salinity of 36. In contrast, growth stimulation of G. carpenteri (strain NQAIF116) was observed at a salinity of 26 and particularly at 16 when co-cultured with Ostreopsis-dominated assemblages. Range expansion of ciguatera-causing dinoflagellates could lead to higher frequency of reported ciguatera illness in populated temperate Australian regions, outside the tropical range of the GBR. Therefore, the findings on salinity and temperature tolerance of two strains of G. carpenteri indicates potential adaptability to different local environmental conditions. These are baseline data for future investigations into the potential southward range expansion of ciguatera-causing dinoflagellates originating from the GBR.     

MORPHOLOGIC DETAILS OF SIX BENTHIC SPECIES OF PROROCENTRUM (PYRROPHYTA) FROM A MANGROVE ISLAND,TWIN CAYS,BELIZE, INCLUDING TWO NEW SPECIES1

Two new dinoflagellate species, Prorocentrum hoffmannianum and Prorocentrum ruetzlerianum, and four known species, Prorocentrum emarginatum Fukuyo 1981, Prorocentrum mesicanum Tafall 1942, Prorocentrum concavum Fukuyo 1981, and Prorocentrum lima (Ehr.) Dodge 1975, from floating detritus and sediments in a subtropical mangrove island, Twin Cays, Belize, Central America are described from scanning electron micrographs. Differences in the following characters of surface micromorphology separated the species: ornamentation of thecal plates (shape, size, and number of valve pores and areolae) and the architecture of the periflagellar area and intercalary band.     

Characterization of Gambierdiscus lapillus sp. nov. (Gonyaulacales,Dinophyceae): a new toxic dinoflagellate from the Great Barrier Reef (Australia)

Gambierdiscus is a genus of benthic dinoflagellates found worldwide. Some species produce neurotoxins (maitotoxins and ciguatoxins) that bioaccumulate and cause ciguatera fish poisoning (CFP), a potentially fatal food‐borne illness that is common worldwide in tropical regions. The investigation of toxigenic species of Gambierdiscus in CFP endemic regions in Australia is necessary as a first step to determine which species of Gambierdiscus are related to CFP cases occurring in this region. In this study, we characterized five strains of Gambierdiscus collected from Heron Island, Australia, a region in which ciguatera is endemic. Clonal cultures were assessed using (i) light microscopy; (ii) scanning electron microscopy; (iii) DNA sequencing based on the nuclear encoded ribosomal 18S and D8‐D10 28S regions; (iv) toxicity via mouse bioassay; and (v) toxin profile as determined by Liquid Chromatography‐Mass Spectrometry. Both the morphological and phylogenetic data indicated that these strains represent a new species of Gambierdiscus, G. lapillus sp. nov. (plate formula Po, 3′, 0a, 7″, 6c, 7‐8s, 5?, 0p, 2″″ and distinctive by size and hatchet‐shaped 2′ plate). Culture extracts were found to be toxic using the mouse bioassay. Using chemical analysis, it was determined that they did not contain maitotoxin (MTX1) or known algal‐derived ciguatoxin analogs (CTX3B, 3C, CTX4A, 4B), but that they contained putative MTX3, and likely other unknown compounds.     

The prevalence of benthic dinoflagellates associated with ciguatera fish poisoning in the central Red Sea

This study confirms the presence of the toxigenic benthic dinoflagellates Gambierdiscus belizeanus and Ostreopsis spp. in the central Red Sea. To our knowledge, this is also the first report of these taxa in coastal waters of Saudi Arabia, indicating the potential occurrence of ciguatera fish poisoning (CFP) in that region. During field investigations carried out in 2012 and 2013, a total of 100 Turbinaria and Halimeda macroalgae samples were collected from coral reefs off the Saudi Arabian coast and examined for the presence of Gambierdiscus and Ostreopsis, two toxigenic dinoflagellate genera commonly observed in coral reef communities around the world. Both Gambierdiscus and Ostreopsis spp. were observed at low densities (<200 cells g⁻¹ wet weight algae). Cell densities of Ostreopsis spp. were significantly higher than Gambierdiscus spp. at most of the sampling sites, and abundances of both genera were negatively correlated with seawater salinity. To assess the potential for ciguatoxicity in this region, several Gambierdiscus isolates were established in culture and examined for species identity and toxicity. All isolates were morphologically and molecularly identified as Gambierdiscus belizeanus. Toxicity analysis of two isolates using the mouse neuroblastoma cell-based assay for ciguatoxins (CTX) confirmed G. belizeanus as a CTX producer, with a maximum toxin content of 6.50 ± 1.14 × 10⁻⁵ pg P-CTX-1 eq. cell⁻¹. Compared to Gambierdiscus isolates from other locations, these were low toxicity strains. The low Gambierdiscus densities observed along with their comparatively low toxin contents may explain why CFP is unidentified and unreported in this region. Nevertheless, the presence of these potentially toxigenic dinoflagellate species at multiple sites in the central Red Sea warrants future study on their possible effects on marine food webs and human health in this region.     

COOLIA CANARIENSIS SP. NOV. (DINOPHYCEAE), A NEW NONTOXIC EPIPHYTIC BENTHIC DINOFLAGELLATE FROM THE CANARY ISLANDS1

A new photosynthetic dinoflagellate species, Coolia canariensis S. Fraga sp. nov., is described based on samples taken from tidal ponds on the rocky shore of the Canary Islands, northeast Atlantic Ocean. Its morphology was studied by LM and SEM. It is almost spherical and has a thick smooth theca with many scattered pores. Plate 1′ is the biggest of the epithecal plates, and 7″ is twice as wide as it is long. Phylogeny inferred from the D1/D2 regions of the LSU nuclear rDNA of three strains of C. canariensis and several strains of other Coolia species, C. monotis, C. sp., showed that C. canariensis strains clustered in a well‐supported clade distinct from the other species. No toxins were detected using mouse bioassay, liquid chromatography with Fluorescence detection (LC‐FLD) or liquid chromatography‐mass spectrometry (LC‐MS). Its pigment composition is of the peridinin type of dinoflagellates. Together with this new species, many other strains of C. monotis from the Atlantic Ocean and Mediterranean Sea have been analyzed for toxin presence, and no evidence of toxin production related to yessotoxins (YTXs) was found, as was previously suggested for C. monotis from Australia.     

Roles of mixotrophy in blooms of different dinoflagellates: Implications from the growth experiment

Studies over the last two decades suggested that mixotrophy could be an important adaptive strategy for some bloom-forming dinoflagellates. In the coastal waters adjacent to the Changjiang River estuary in the East China Sea, recurrent blooms of dinoflagellates Prorocentrum donghaiense, Karenia mikimotoi and Alexandrium catenella started to appear from the beginning of the 21 century, but roles of mixotrophy in the formation of dinoflagellate blooms were not well understood. In the current study, mixotrophy-based growth of four selected bloom-causative dinoflagellate species, i.e. K. mikimotoi, A. catenella, P. donghaiense and Prorocentrum micans, were studied. Dinoflagellates were co-cultured with different prey organisms, including bacterium Marinobacter sp., microalgae Isochrysis galbana and Hemiselmis virescens, under a variant of nutrient conditions. It was found that growth of dinoflagellate K. mikimotoi was significantly promoted with the presence of prey organisms. Growth of P. donghaiense and P. micans was only slightly improved. For A. catenella, the addition of prey organisms has no effects on the growth, while both of the two prey microalgae I. galbana and H. virescens were killed, probably by allelochemicals released from A. catenella. There was no apparent relationship between nutrient conditions and the mixotrophy-based growth of the tested dinoflagellates. Based on the results of the growth experiment, it is implicated that mixotrophy may play different roles in the growth and bloom of the four dinoflagellate species. It can be an important competitive strategy for K. mikimotoi. For the two Prorocentrum species and A. catenella, however, the role of mixotrophy is much limited. They may depend more on other competitive strategies, such as phototrophy-based growth and allelopathic effect, to prevail in the phytoplankton community and form blooms.     

Revised dinoflagellate phylogeny inferred from molecular analysis of large-subunit ribosomal RNA gene sequences

The nucleotide sequence analysis of the PCR products corresponding to the variable large-subunit rRNA domains D1, D2, D9, and D10 from ten representative dinoflagellate species is reported. Species were selected among the main laboratory-grown dinoflagellate groups: Prorocentrales, Gymnodiniales, and Peridiniales which comprise a variety of morphological and ecological characteristics. The sequence alignments comprising up to 1,000 nucleotides from all ten species were employed to analyze the phylogenetic relationships among these dinoflagellates. Maximum parsimony and neighbor joining trees were inferred from the data generated and subsequently tested by bootstrapping. Both the D1/D2 and the D9/D10 regions led to coherent trees in which the main class of dinoflagellates, Dinophyceae, is divided in three groups: prorocentroid, gymnodinioid, and peridinioid. An interesting outcome from the molecular phylogeny obtained was the uncertain emergence of Prorocentrum lima. The molecular results reported agreed with morphological classifications within Peridiniales but not with those of Prorocentrales and Gymnodiniales. Additionally, the sequence comparison analysis provided strong evidence to suggest that Alexandrium minutum and Alexandrium lusitanicum were synonymous species given the identical sequence they shared. Moreover, clone Gg1V, which was determined Gymnodinium catenatum based on morphological criteria, would correspond to a new species of the genus Gymnodinium as its sequence clearly differed from that obtained in G. catenatum. The sequence of the amplified fragments was demonstrated to be a valuable tool for phylogenetic and taxonomical analysis among these highly diversified species. Correspondence to: J. M. Bautista     

Determination of diarrhetic shellfish toxins in various dinoflagellate species

Sixteen species of unialgal samples of dinoflagellate, either wild or cultured, were tested for production of diarrhetic shellfish toxins such as okadaic acid (OA), dinophysistoxin-1 (DTX1), and pectenotoxins (PTXs). Determination of micro-quantities of the toxins was facilitated by fluorometry and UV HPLC. Seven Dinophysis species were confirmed to produce either OA or DTX1, or both. Toxin content and composition varied regionally and seasonally. Intraspecies variation was also observed among cultured strains of Prorocentrum lima. PTX2 was the only toxin detected among PTX family, and D. fortii was the only species to contain this toxin. author for correspondence     

Are crustaceans linked to the ciguatera food chain?

Synopsis Adult brine shrimp, Artemia spp., were used as an experimental organism to elucidate the role that crustacea may play in the transference of ciguatera toxins. Some ciguatera-implicated dinoflagellates were highly toxic to brine shrimp that had consumed them. Four clones of Gambierdiscus toxicus were fed in four trials at rates ranging from 2 to 480 cells per adult brine shrimp; the 24 h LD₅₀ for the four clones were 2.8, 33.4, 41.1, and 104.5 cells per brine shrimp. Dinoflagellates Prorocentrum concavum and P. lima were also fed to adult brine shrimp, but minimal mortalities occurred at cell concentrations ranging up to 1000 cells per test animal. Tilapine cichlid (Oreochromis niloticus ×O. mossambicus) young fed brine shrimp containing G. toxicus cells displayed behavioral abnormalities ranging from spiral swimming to loss of equilibrium. The present data suggest that toxins accumulated by dinoflagellate-consuming crustaceans could produce toxicity in zooplanktivorous fish species, or to detritivores in cases where dinoflagellate consumption resulted in crustacean mortalities. Field studies of the ciguatera food chain should be expanded to include examination of crustacean diets to more fully define their role in toxin transfer.     

Inhibition of dinoflagellate growth by extracts of barley straw (Hordeum vulgare)

Growth of dinoflagellates representing three orders, the Gymnodiniales,Peridiniales, and the Prorocentrales was examined following treatment withbarley straw extract. Selected dinoflagellate taxa showed growth responsessimilar to those reported for freshwater algae including: inhibition(Gyrodinium galatheanum, Gymnodiniumsanguineum, Heterocapsa triquetra andH. pygmaea); stimulation (Gyrodinium instriatum,Prorocentrum minimum and P. micans); and no effect(Gyrodinium estuariale, G. uncatenum,Ceratium furca, Peridinium sp.).Although barley straw extracts do not appear to have value as a universalmanagement tool for dinoflagellates, they may have potential in management ofspecific taxa and possibly taxonomic groups.     

Effects of temperature,salinity and their interaction on growth of the benthic dinoflagellate Ostreopsis cf. ovata (Dinophyceae) from Japanese coastal waters

Benthic dinoflagellates of the genus Ostreopsis produce palytoxin (PTX)‐like compounds. The worldwide distributed Ostreopsis ovata/O. cf. ovata is potentially responsible for outbreaks of human health problems around the coasts of tropical, subtropical, and temperate regions. The present study examined growth responses of an O. cf. ovata strain s0662 collected from coastal waters of Japan with 35 different combinations of temperature (15–35°C) and salinity (20–40) and discusses the bloom dynamics of the organism in Japanese coastal environments. The O. cf. ovata strain s0662 tolerated a wide range of temperature (17.5–35°C) and salinity (25–40). Results of a two‐way ANOVA showed significant effects of temperature‐salinity interaction on growth rates and biomass yields of the O. cf. ovata strain (F(24,70) > 127, P < 0.001). The strain showed a maximal growth rate (1.03 divisions day^?1) and biomass yield (240 relative fluorescence) at temperature 25°C and salinity 30. The high growth rates of over 1.0 division day^?1 were obtained in conditions of temperature 25–30°C and salinity 30–35, which indicates that strain s0662 prefers high temperature and salinity conditions. The growth rates of O. cf. ovata under the optimal conditions were higher than those of other benthic toxic‐dinoflagellates, Coolia monotis, Gambierdiscus toxicus, and Prorocentrum lima (Dinophyceae) previously reported. Taken together, we suggest that O. cf. ovata is able to grow faster than the other benthic dinoflagellates in waters of high temperature and salinity. The physiological feature probably confers an ecological advantage on O. cf. ovata in the bloom development during warmer seasons in Japan and may be responsible for outbreaks of PTX‐like poisoning in the region especially during the warmer seasons.     

Morphological and genetic analysis of the Coolia monotis species complex with the introduction of two new species,Coolia santacroce sp. nov. and Coolia palmyrensis sp. nov. (Dinophyceae)

The dinoflagellate genus Coolia Meunier is an important epi-benthic organism that is commonly found in association with other dinoflagellates known to cause ciguatera. Two closely related taxa, Coolia monotis and Coolia malayensis, make up the C. monotis species complex. In this study we introduce two new toxic species that should be included in that complex, Coolia palmyrensis Karafas, Tomas, York sp. nov. and Coolia santacroce Karafas, Tomas, York sp. nov., collected from the Palmyra Atoll in the Pacific Ocean and Saint Croix, US Virgin Islands, respectively. These two species can be distinguished morphologically by size, pore shape, pore density, and the relative size of the apical pore complex. The ITS1/5.8S/ITS2 and the D1/D2 regions of the LSU rDNA were used to provide molecular support of morphological observations using maximum likelihood and Bayesian analyses. Furthermore, C. palmyrensis and C. santacroce both showed cytotoxic effects on human derived cells in vitro.