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As historically described, the genus Amphidinum has long been recognized to be polyphyletic. Amphidinium sensu lato is the most diverse of all marine benthic dinoflagellate genera and after the redefinition of the sensu stricto genus about 100 species remained of unknown generic affiliation. A species similar to the Australian sensu lato species Amphidinium boggayum was discovered in France. Morphological and molecular phylogenetic data supported the description of a new species and the classification of the two closely related species within a new athecate, photosynthetic, benthic genus, named Bindiferia. Cells were dorsoventrally flattened with smaller asymmetric epicone. The cingulum was descending. The sulcus reached the antapex and had an extension onto the epicone. The apical structure complex formed an anticlockwise loop around the apex. A red stigma was located in the anterior part of the epicone, near the apex. Vegetative division occurred in a non-motile stage within a hyaline sheath. Non-motile cells had dorsal hypocone grooves. The two species differed from each other in epicone size, cingulum displacement, nucleus location and surface groove occurrence. The molecular phylogenetic results showed that Bindiferia is a distinct taxon that is only distantly related to Amphidinium sensu stricto. The nearest sister group to Bindiferia could not be reliably determined, but the apical structure complex suggested a close relationship to the genera of the Gymnodinium sensu stricto clade.

Gymnodinium gracile, described from the coasts of Denmark in 1881, is one of the first described unarmored dinoflagellates. Individuals which morphologically fit with the original description were isolated from the English Channel (North‐East Atlantic). The SSU rRNA gene sequences were identical to the sequences identified as Balechina pachydermata and Gymnodinium amphora from the Mediterranean Sea and Brazil. We propose the transfer of Gymnodinium gracile into the genus Balechina as B. gracilis comb. nov. These sequences constitute an independent lineage, clustering with numerous environmental sequences from polar to tropical waters. The widespread distribution, the high plasticity in size, shape and coloration and the difficulties in discerning the fine longitudinal striae have contributed to the description of numerous synonyms: Amphidinium vasculum, Balechina pachydermata (=Gymnodinium pachydermatum), Gymnodinium achromaticum, G. abbreviatum, G. amphora, G. dogielii, G. lohmannii (=G. roseum sensu Lohmann 1908), G. situla and Gyrodinium cuneatum (=G. gracile sensu Pouchet 1885).

There is growing interest in finding microalgae species that efficiently convert dissolved nutrients contained in aquaculture effluents into highly valuable biomass. The different nitrogen forms that are present in aquaculture effluents are particularly concerning. This study demonstrated that the dinoflagellate Amphidinium carterae can acclimate to both combined and sole nitrogen sources such as nitrate, ammonium, and urea over a wide concentration range. As far as is known, it is the first time that a species of the genus Amphidinium has been successfully cultured with urea as the sole source of nitrogen. In the presence of 882 μM of nitrate, A. carterae tolerated urea concentrations up to 5000 μM. With respect to ammonium-N tolerance, it has been observed that it is lethal at concentrations higher than 441 μM. A robust laboratory experimental design was critical for accurately assessing this acclimation. Alternative N sources did not affect the production of high-value specific polyketide secondary metabolites from A. carterae, such as amphidinols, with an average concentration of 0.435 ± 0.038% biomass d.w. An analysis of the symbiotic microbial assemblages developed in a long-term A. carterae culture in an open raceway pond, and the fact that it is able to metabolize all three nitrogen sources simultaneously, supports the idea that this microalga has the potential to be successfully cultured with aquaculture effluents.

The present study investigated selected benthic dinoflagellates isolated from different regions from the Western Atlantic, with respect to their morphology, growth, toxicity, and toxin production in culture. A total of nine strains of benthic dinoflagellates belonging to three genera were cultivated: Amphidinium massartii, Amphidinium operculatum (2 strains), Coolia malayensis (2 strains), Prorocentrum hoffmannianum (2 strains), and Prorocentrum mexicanum, whose morphological and genetic characterizations matched previous descriptions, and Prorocentrum sp., morphologically related to P. cf. norrissianum. The two strains of C. malayensis from Brazil attained the highest growth rates (0.42–0.47 day−1), but the lowest cell densities (2.2–2.9 × 104 cells mL−1) in culture. The highest cell densities were recorded for A. massartii from Cuba (3.8 × 105 cells mL−1). All species/strains investigated exhibited moderate toxicity to larvae of the brine shrimp Artemia salina; A. massartii being the most toxic species and Prorocentrum sp. the least one. Additionally, extracts of Prorocentrum species (P. hoffmannianum and Prorocentrum sp.) tested positive in mouse bioassays following intraperitoneal injection. Moderate to high concentrations of okadaic acid (OA), but no dinophysistoxins (DTXs), were found in both P. hoffmannianum strains from Cuba; but no diarrheic toxins were detected in either P. mexicanum from Cuba or Prorocentrum sp. from southern Brazil. Finally, five novel amphidinols were detected in cultures of both A. massartii (Cuba) and A. operculatum (Brazil) by LC-MS/MS, with molecular weights of 1440.8 (two isomers), 1360.8, 1287.7, and 984.6. These findings clearly indicate the need to include benthic species among the harmful microalgae surveyed in regional monitoring programs of phytoplankton.

Background Some dinoflagellates cause harmful algal blooms, releasing toxic secondary metabolites, to the detriment of marine ecosystems and human health. Our understanding of dinoflagellate toxin biosynthesis has been hampered by their unusually large genomes. To overcome this challenge, for the first time, we sequenced the genome, microRNAs, and mRNA isoforms of a basal dinoflagellate, Amphidinium gibbosum, and employed an integrated omics approach to understand its secondary metabolite biosynthesis. Results We assembled the ~ 6.4-Gb A. gibbosum genome, and by probing decoded dinoflagellate genomes and transcriptomes, we identified the non-ribosomal peptide synthetase adenylation domain as essential for generation of specialized metabolites. Upon starving the cells of phosphate and nitrogen, we observed pronounced shifts in metabolite biosynthesis, suggestive of post-transcriptional regulation by microRNAs. Using Iso-Seq and RNA-seq data, we found that alternative splicing and polycistronic expression generate different transcripts for secondary metabolism. Conclusions Our genomic findings suggest intricate integration of various metabolic enzymes that function iteratively to synthesize metabolites, providing mechanistic insights into how dinoflagellates synthesize secondary metabolites, depending upon nutrient availability. This study provides insights into toxin production associated with dinoflagellate blooms. The genome of this basal dinoflagellate provides important clues about dinoflagellate evolution and overcomes the large genome size, which has been a challenge previously.

Eukaryotic microalgae have developed CO2concentrating mechanisms to maximise the concentration of CO2 at the active site of Rubisco in response to the low CO2 concentrations in the external aquatic medium. In these organisms, the modes of inorganic carbon (Ci) uptake are diverse, ranging from diffusive CO2 uptake to the active transport of HCO3 -and CO2 and many have an external carbonic anhydrase to facilitate HCO3- use. There is unequivocal evidence for the mechanisms of Ci uptake in only about 25 species of microalgae of the chlorophyte, haptophyte, rhodophyte, diatom, and eustigmatophyte groups. Most of these species take up both CO2 and HCO3-, but the rates of uptake of each of these substrates varies with the algal species. A few species take up only one of the two forms of Ci, an adaptation that is not necessarily correlated with their ecological distribution. Evidence is presented for the active uptake of HCO3- and CO2 in two marine haptophytes,Isochrysis galbana Parke and Dicrateria inornata Parke, and for active transport of CO2 but lack of HCO3- uptake in two marine dinoflagellates, Amphidinium carteraeHulburt and Heterocapsa oceanica Stein.

For the first time, distribution of epiphytic dinoflagellates was studied in Saint Martin Island (Lesser Antilles) during the cyclonic season (between September 1st and 3rd, 2015). The present study provides a semi-quantitative analysis because the fresh weight of each macrophyte was estimated around 10 g. The identified genera were: Ostreopsis J.Schmidt, Prorocentrum Ehrenberg, Coolia Meunier, Amphidinium Claperede & Lachmann, and Gambierdiscus Adachi & Fukuyo in order of decreasing abundance. Highest average abundance values of the genera Ostreopsis and Amphidinium were hosted by macrophytes of the Phaeophyceae class with c. 15 000 and 60 cells g–1 respectively. Epiphytic Coolia cells were mainly observed on seagrasses with the highest average abundance value of c. 1000 cells g–1 whereas the genera Gambierdiscus and Prorocentrum were most often associated with Florideophyceae with the highest average abundances of c. 70 and 1500 cells g–1 respectively. This preliminary study indicates the most relevant locations to survey the biodiversity of potentially toxic epiphytic dinoflagellates in Saint Martin Island.

Ciguatera fish poisoning (CFP) is prevalent around the tropical and sub-tropical latitudes of the world and impacts many Pacific island communities intrinsically linked to the reef system for sustenance and trade. While the genus Gambierdiscus has been linked with CFP, it is commonly found on tropical reef systems in microalgal assemblages with other genera of toxin-producing, epiphytic and/or benthic dinoflagellates – Amphidinium, Coolia, Fukuyoa, Ostreopsis and Prorocentrum. Identifying a biomarker compound that can be used for the early detection of Gambierdiscus blooms, specifically in a mixed microalgal community, is paramount in enabling the development of management and mitigation strategies. Following on from the recent structural elucidation of 44-methylgambierone, its potential to contribute to CFP intoxication events and applicability as a biomarker compound for Gambierdiscus spp. was investigated. The acute toxicity of this secondary metabolite was determined by intraperitoneal injection using mice, which showed it to be of low toxicity, with an LD50 between 20 and 38 mg kg−1. The production of 44-methylgambierone by 252 marine microalgal isolates consisting of 90 species from 32 genera across seven classes, was assessed by liquid chromatography-tandem mass spectrometry. It was discovered that the production of this secondary metabolite was ubiquitous to the eight Gambierdiscus species tested, however not all isolates of G. carpenteri, and some species/isolates of Coolia and Fukuyoa.

The genus Amphidinium is an important group of athecated dinoflagellates because of its high abundance in marine habitats, its member's ability to live in a variety of environmental conditions and ability to produce toxins. Furthermore, the genus is of particular interest in the biotechnology field for its potential in the pharmaceutical arena. Taxonomically the there is a history of complication and confusion over the proper identities and placements of Amphidinium species due to high genetic variability coupled with high morphological conservation. Thirteen years has passed since the most recent review of the group, and while many issues were resolved, some remain. The present study used microscopy, phylogenetics of the 28S region of rDNA, secondary structure of the ITS2 region of rDNA, compensatory base change data, and cytotoxicity data from Amphidinium strains collected world-wide to elucidate remaining confusion. This holistic approach using multiple lines of evidence resulted in a more comprehensive understanding of the morphological, ecological, and genetic characteristics that are attributed to organisms belonging to Amphidinium, including six novel species: A. fijiensis, A. magnum, A. paucianulatum, A. pseudomassartii, A. theodori, and A. tomasii.

A new marine benthic dinoflagellate, Testudodinium magnum sp. nov., was described from a sand sample collected on the seafloor at a depth of 35 m off Mageshima Island, Kagoshima, Japan. The dinoflagellate possessed the characteristic features of the genus Testudodinium, but was distinguished by its extremely large size and pebbled dorsal surface of the hyposome, caused by the presence of numerous nodules. The cells of this dinoflagellate possessed a dominant sessile and a rare motile form that were morphologically distinct from each other: The sessile form was larger and the small rounded episome with longitudinal furrow was completely embedded in the hyposome; the motile form was smaller and the episome was only partly embedded in the hyposome. The ultrastructural investigations revealed the presence of a large, circular, starch-sheathed pyrenoid whose matrix was traversed by randomly arranged thylakoid lamellae. The chloroplasts formed a network radiating outward from the pyrenoid. Uniquely, t...

Climate driven increases in ocean temperature and pCO2 have the potential to alter the growth and prevalence of future Harmful Algal Blooms (HABs), but systematic studies on how climate drivers influence toxic algal species relative to non-toxic phytoplankton are lacking. In particular, little is known about how future climate scenarios will affect the growth of the toxic dinoflagellate Alexandrium catenella, which is responsible for the paralytic shellfish poisoning (PSP) events that threaten the health and economy of coastal communities in the Gulf of Maine and elsewhere. The growth responses of A. catenella and two other naturally co-occurring dinoflagellates in the Gulf of Maine—Scrippsiella sp., and Amphidinium carterae—were studied in mono and mixed species cultures. Experimental treatments tested the effects of elevated temperature (20 °C), lower pH (7.8), and the combination of elevated temperature and lower pH on growth rates relative to those in near-current conditions (15 °C; pH 8.1). Growth rates of A. catenella decreased under elevated temperature and lower pH conditions, a response that was largely attributable to the effect of temperature. In contrast, growth rates of Scrippsiella sp. and A. carterae increased under elevated temperature and lower pH conditions, with temperature also being the primary driver of the response. These trends did not change substantially when these species were grown in mixed cultures (A. catenella + Scrippsiella sp., and A. catenella + A. carterae), indicating that allelopathic or competitive interactions did not affect the experimental outcome under the conditions tested. These findings suggest that A. catenella blooms may become less prevalent in the southern regions of the Gulf of Maine, but potentially more prevalent in the northeastern regions of the Gulf of Maine with continued climate change.

One of the observed flagellates, Cryptella cyanophora Pascher, is probably the first discovery ever of this species with cyanelles after more than 80 years since its description. This species was previously classified into the cryptophycean genus Chroomonas Hansgirg (see Discussion), which is why the observations of two common species, Chroomonas nordstedtii Hansg. and Ch. caudata Geitler, from natural water bodies are also presented and the theory of their cytological evolution is discussed. Similar uncertainty is associated with the origin of blue-green chloroplasts of some dinoflagellates, two species of which (from Czech waters) are presented, namely Gymnodinium aeruginosum Stein and Amphidinium amphidinioides (Geitler) Schiller. Again the theory of the origin of their chloroplast is discussed. The indubitable syncyanosis is demonstrated on Glaucocystis nostochinearum Itzigsohn.

Marine microalgae and cyanobacteria have largely been studied for their biotechnological potential and proved their ability to produce a wide array of bioactive molecules. We investigated the antifouling potential of unexplored benthic tropical microalgae using anti-adhesion and toxicity bioassays against two major micro- and ma crobiofoulers, namely bacteria and barnacles. Fifty strains belonging to six phyla [Cyanobacteria, Miozoa (Dinoflagellata), Bacillariophyta, Cryptophyta, Rhodophyta and Haptophyta] were isolated from southwestern Islands of the Indian Ocean. They were chosen in order to represent as much as possible the huge biodiversity of such a rich tropical ecosystem. The associated chemodiversity was highlighted by both NMR- and LC-MS-based metabolomics. The screening of 84 algal fractions revealed that the anti-adhesion activity was concentrated in methanolic ones (i.e. 93% of all active fractions). Our results confirmed that microalgae constitute a promising source of natural antimicrofoulants as 17 out of the 30 active fractions showed high or very high capacity to inhibit the adhesion of three biofilm-forming marine bacteria. Dinoflagellate-derived fractions were the most active, both in terms of number and intensity. However, dinoflagellates were also more toxic and may not be suitable as a source of environmentally friendly antifouling compounds, in contrast to diatoms, e.g. Navicula mollis. The latter and two dinoflagellates of the genus Amphidinium also had interesting anti-settlement activities while being moderately toxic to barnacle larvae. Our approach, combining the bioprospecting of a large number of tropical microalgae for their anti-settlement potential and metabolomics analyses, constituted a first step towards the discovery of alternative ecofriendly antifoulants.

The so-called unarmoured dinoflagellates are not a “natural” (phylogenetic) group but they lack thecal plates, share fragility and possess relatively few morphological characters that can be positively identified. This study depicts the species composition of unarmoured dinoflagellates collected from sites along the coasts of the Gulf of Mexico and the Mexican Pacific and includes their descriptions and illustrations. We identified a total of 25 species belonging to 13 genera and six families that were studied through various techniques using light and scanning electron microscopy. Seven new records for the Mexican Pacific are annotated here that include Amphidinium flagelans Schiller, Gyrodinium cochlea Lebour, G. glaebum Hulburt, G. metum Hulburt, Karenia selliformis Haywood, Steidinger et MacKenzie, Karlodinium ballantinum de Salas, and K. veneficum (Ballantine) Larsen. The genera Karenia and Karlodinium belong to the family Kareniaceae, characterised for encompassing the largest number of toxic species among the unarmoured dinoflagellates, and many species produce algal blooms around the world. Ceratoperidinium falcatum and Levanderina fissa are names recently proposed elsewhere as new taxonomic combinations and are also included here. The diversity of unarmoured dinoflagellates has been strongly underestimated in the past and the new records reported here confirm this. Further studies, including traditional and modern concepts and protocols (including molecular tools), should be undertaken in the near future to understand their real diversity.

Se estudió a los dinoflagelados epífitos en 250 muestras de 10 sitios en Chelem (una laguna rodeada parcialmente por manglar) y Dzilam de Bravo (una localidad costera expuesta), en la costa norte de la Península de Yucatán, obtenidas durante cinco exploraciones en 2008-2009. Se midió la temperatura, salinidad, turbidez, pH, oxígeno disuelto, nitratos, nitritos, fosfatos, silicatos, urea, clorofila-a extraída de la columna de agua, precipitación, así como velocidad y dirección del viento. El sistema de la laguna de Chelem reveló menor variabilidad de las características físico-químicas comparado con la localidad expuesta de Dzilam de Bravo. Los dinoflagelados se encontraron asociados con todos los macrófitos hospederos incluyendo cuatro especies de pastos marinos y 33 de macroalgas representantes de 24 géneros. Un total de 20 taxa de dinoflagelados correspondientes a 12 géneros se obtuvieron de estos sustratos. El género Prorocentrum incluyó la mayor cantidad de especies. La variación en la abundancia promedio de dinoflagelados epifíticos en ambas localidades osciló entre ~200 y 3500 células g-1 de peso húmedo de sustrato. La abundancia de células en los sitios, en contraste, varió entre ~100 y >25 000 células g-1. Esta variación es típica de la distribución en parches de estas especies en el tiempo y el espacio. En general, Prorocentrum rhathymum (máximo 2.41x104 células g-1) fue la especie más abundante en las muestras. Otras especies abundantes fueron Bysmatrum caponii (máximo 1.19x104 células g-1) y Amphidinium cf. carterae (máximo 3.69x103 células g-1). La abundancia máxima deGambierdiscus spp. se presentó en mayo y noviembre (9.90x103 células g-1) en Chelem cuando la temperatura varió de 24.5 a 30.2 °C. Los datos obtenidos muestran que el mayor potencial para el flujo de ciguatoxinas a través de la red trófica puede encontrarse en ambientes protegidos de turbulencia baja, donde la salinidad es alta, los nutrientes abundantes y la temperatura de agua entre 24 y 31 oC.

Macroalgae growing in New Zealand's sub-tropical waters were sampled for epiphytic microalgae, in particular dinoflagellates. Four new Coolia isolates collected from sites throughout Northland in January/February 2013 were all identified as C. malayensis as determined by large subunit ribosomal DNA (LSU rDNA) sequence analysis and scanning electron microscopy. C. malayensis , a common dinoflagellate species in New Zealand, was previously reported as C. monotis and isolates held in the Cawthron Institute Culture Collection of Microalgae have now been reclassified based on the DNA sequence data. Toxicity studies of the New Zealand C. malayensis isolates (as determined by intraperitoneal (i.p.) injection of mice) resulted in an LD 50 of 80.0 mg/kg for isolate NLD 12 (95% CI: 63.8–101.0 mg/kg) compared to a Malaysian isolate which exhibited low toxicity, with transient effects at 900 mg/kg, but which resulted in the enlargement of the test animal's spleen. Ostreopsis siamensis , a common epiphytic bloom former in northern New Zealand, was found in association with Coolia at sites across Northland. Densest cell concentrations were collected from macroalgae throughout the Bay of Islands. Four new isolates of O. siamensis all produced palytoxin-like compounds (LC–MS analysis). Also isolated from Northland macroalgae were Amphidinium thermaeum (non-toxic by i.p. injection of mice) and Gambierdiscus cf. yasumotoi , with the latter producing a putative maitotoxin analogue (MTX-3) as determined by LC–MS.

Ciguatera is a serious seafood poisoning syndrome caused by the consumption of ciguatoxin-contaminated finfish from tropical and subtropical regions. This study examined the community structure of ciguatera-associated dinoflagellates and the distribution pattern, taxonomy and toxicity of Gambierdiscus spp . from a high-risk area of Marakei, Republic of Kiribati. The genera Gambierdiscus , Prorocentrum , Ostreopsis , Amphidinium and Coolia were present, and generally the former three dominated the dinoflagellate assemblage. Among these three, Gambierdiscus was the most abundant dinoflagellate genus observed at three of the four sites sampled, two of which (Sites 1 and 2) were on the northern half of the island and two (Sites 3 and 4) on the southern half. The following patterns of abundance were observed among sites: (1) Average Gambierdiscus spp. abundance at the northern sites exceeded the southern sites by a factor of 19–54; and (2) Gambierdiscus spp. abundance at shallow sites (2–3 m) exceeded deeper sites (10–15 m). The distribution of Gambierdiscus spp. at Marakei corresponded with previously observed patterns of fish toxicity, with fish from southern locations being much less toxic than fish sampled north of the central channel. DNA sequencing identified three Gambierdiscus species ( Gambierdiscus carpenteri , Gambierdiscus belizeanus , Gambierdiscus pacificus ) and three previously unreported ribotypes ( Gambierdiscus sp. type 4, Gambierdiscus sp. type 5, Gambierdiscus sp. type 6) in the samples; Gambierdiscus sp. type 4 may represent a Pacific clade of Gambierdiscus sp. ribotype 1. Toxicity analyses determined that Gambierdiscus sp . type 4 isolates were more toxic than the Gambierdiscus sp . type 5 and G. pacificus isolates, with toxin contents of 2.6–6.0 (mean: 4.3 ± 1.4), 0.010 and 0.011 fg P-CTX-1 eq cell −1 , respectively. Despite low densities of Gambierdiscus spp. observed at Marakei relative to other studies in other parts of the world, the presence of low and moderately toxic populations may be sufficient to render the western coast of Marakei a high-risk area for ciguatera. The long history of toxicity along the western side of Marakei suggests that large-scale oceanographic forcings that regulate the distribution of Gambierdiscus spp. along the western side of Marakei may have remained relatively stable over that time. Chronic as well as acute exposure to ciguatoxins may therefore pose an important human health impact to the residents of Marakei.

A new dinoflagellate species (Dinophyceae, Gymnodiniales)—Apicoporus haificum Krachmalny sp. nov. was described on a basis of thorough light microscopy studies. The species was found in sandy sediments (water-filled spaces between sand grains) on the eastern coast of the Mediterranean Sea (HaCarmel beach, Haifa, Israel). Apicoporus haificum possesses the following features: cells elongated (with length exceeding width by 3.1–3.8 times), lanceolate in dorsoventral projection, roundish in cross-section with slight dorsoventral compression, asymmetrical, slightly curved to the right, with antapical depression. Epicone asymmetrical, somewhat umbonate, with a small hook-shaped protrusion on the apex. Cingulum displaced, descending by 1/3 cell’s length, its distal part almost parallel to the anterior portion of sulcus. Sulcus narrow, shallow, extends from the apex to the antapical depression, with apical groove. Cell surface covered with longitudinal striations that converge on the apex and on the antapical protrusion. Chloroplasts absent. Dimensions: 75.3±2.2 μm in length, 21.4±1.5 μm in width. Comparison of A. haificum to morphologically similar Apicoporus glaber and Amphidinium scissum is discussed.

Summary Peridinin-containing dinoflagellates have small circular DNA molecules called minicircle DNAs, each of which encodes one, or occasionally a few, plastid proteins or ribosomal RNA. Dinoflagellate minicircle DNA is composed of two parts: a gene-coding sequence and a non-coding sequence that consists of several variable and core regions. The core regions are identical among the minicircle DNAs with different genes within a species or strain. Because such structure is very different from those of well known plastid DNAs, many functional and evolutionary questions have been raised for the minicircle DNAs, and several studies that focus on answering those questions are underway. However, the localization of minicircle DNA is still controversial: several lines of indirect evidence have implied plastid localization, whereas the nuclear localization of minicircle DNA has also been suggested in a species. In order to understand the evolution and function of minicircle DNA, it is important to know its precise localization. In this study, we sequenced two typical minicircle DNAs, one encodes psbA and the other encodes 23S rRNA genes, from an Amphidinium massartii strain (TM16). To determine the subcellular localization of these minicircle DNAs, we performed DNA-targeted whole cell fluorescence in situ hybridization with A. massartii minicircle DNA-specific probes and demonstrated that minicircle DNAs were present in plastids. This study provides the first direct evidence for the plastid localization of dinoflagellate minicircle DNAs.

Amphidinium massartii Biecheler is an epiphytic and toxic dinoflagellate. Prior to the present study, A. massartii has been reported in the waters off the Mediterranean, Australian, USA, and Canadian coasts. We isolated Amphidinium cells from the coastal waters of Jeju Island, Korea and their morphology and rDNA sequences indicated that they were A. massartii. Herein, we report for the first time the occurrence of A. massartii in the waters of the temperate region in the northwestern Pacific Ocean. The large subunit (LSU) rDNA sequences of the Korean strains were 0.7% different from those of an Australian strain of A. massartii CS-259, the closest species, but were 4.1-5.8% different from those of the other Australian strains and the USA strains of A. massartii and from those of Amphidinium sp. HG115 that was isolated from subtropical Okinawan waters. In phylogenetic trees based on LSU, internal transcribed spacer, small subunit rDNA, and cytochrome b sequences, the Korean strains belonged to the A. massartii clade, which was clearly divergent from the A. carterae clade. The morphology of the Korean A. massartii strains was similar to that of the originally described French strain and recently described Australian strain. However, we report for the first time here that scales were observed on the surface of the flagella. In conclusion, the Korean A. massartii strains have unique rDNA sequences, even though they have a very similar morphology to that of previously reported strains. This report extends the known range of this dinoflagellate to the temperate waters of the northwestern Pacific Ocean.

A new athecate dinoflagellate, Bispinodinium angelaceum N. Yamada et Horiguchi gen. et sp. nov., is described from a sand sample collected on the seafloor at a depth of 36 m off Mageshima Island, subtropical Japan. The dinoflagellate is dorsiventrally compressed and axi-symmetric along the sulcus. The morphology resembles that of the genus Amphidinium sensu lato by having a small epicone that is less than one third of the total cell length. However, it has a new type of apical groove, the path of which traces the outline of a magnifying glass. The circular component of this path forms a complete circle in the center of the epicone and the straight "handle" runs from the sulcus to the circular component. Inside the cell, a pair of elongated fibrous structure termed here the "spinoid apparatus" extends from just beneath the circular apical groove to a point near the nucleus. Each of two paired structures consists of at least 10 hyaline fibers and this is a novel structure found in dinoflagellates. Phylogenetic analyses based on the SSU and LSU RNA genes did not show any high bootstrap affinities with currently known athecate dinoflagellates. On the basis of its novel morphological features and molecular signal, we conclude that this dinoflagellate should be described as a new species belonging to a new genus.

The classical athecate dinoflagellate genera (Amphidinium, Gymnodinium, Gyrodinium) have long been recognized to be polyphyletic. Amphidinium sensu lato is the most diverse of all marine benthic dinoflagellate genera; however, following the redefinition of this genus ∼100 species remain now of uncertain or unknown generic affiliation. In an effort to improve our taxonomic and phylogenetic understanding of one of these species, namely Amphidinium semilunatum, we re-investigated organisms from several distant sites around the world using light and scanning electron microscopy and molecular phylogenetic methods. Our results enabled us to describe this species within a new heterotrophic genus, Ankistrodinium. Cells of A. semilunatum were strongly laterally flattened, rounded-quadrangular to oval in lateral view, and possessed a small asymmetrical epicone. The sulcus was wide and characteristically deeply incised on the hypocone running around the antapex and reaching the dorsal side. The straight acrobase with hook-shaped end started at the sulcal extension and continued onto the epicone. The molecular phylogenetic results clearly showed that A. semilunatum is a distinct taxon and is only distantly related to species within the genus Amphidinium sensu stricto. The nearest sister group to Ankistrodinium could not be reliably determined.

SUMMARY A new genus of sand-dwelling photosynthetic dinoflagellate, Testudodinium Horiguchi, Tamura, Katsumata et A. Yamaguchi is proposed based on Testudodinium testudo (Herdman) Horiguchi, Tamura, Katsumata, et A. Yamaguchi comb. nov. (Basionym: Amphidinium testudo Herdman) and a new species in this new genus, Testudodinium maedaense Katsumata et Horiguchi sp. nov. is described. Amphidinium corrugatum is also transferred to this genus, making a new combination T. corrugatum (Larsen et Patterson) Horiguchi, Tamura et A. Yamaguchi. These three species are similar to the members of the genus Amphidinium in having an extremely small episome and a dorsoventrally flattened cell body. They are, however, distinguished from the genus Amphidinium seusu stricto by the possession of a distinct longitudinal furrow in the middle of ventral side of the episome. Phylogenetic trees based on small subunit (SSU) rDNA revealed that all three of these Testudodinium species formed a robust clade and, although statistical support is not high, the tree suggests Testudodinium clade is not closely related to Amphidinium seusu stricto clade. The morphological differences together with molecular data support the establishment of a new genus for A. testudo and its related species.

SUMMARY A new species of Amphidinium, A. cupulatisquama Tamura et Horiguchi, from sand samples from Ikei Island, Okinawa Prefecture in subtropical Japan, is described based on light, scanning and transmission electron microscopy and the partial sequencing of the large subunit rDNA gene. The species has a typical morphology for the genus, but is distinguished from previously described species by having a combination of the following characteristics: (i) a relatively large cell (over 30 µm in length); (ii) possessing an eyespot on the dorsal side of the cingulum; (iii) the longitudinal flagellum emerging from a point close to the cingulum; (iv) cell division taking place in the motile phase; and (v) possessing body scales. This is the third species of this genus to possess body scales. The body scales of A. cupulatisquama are uniform and cup-shaped in side view and elliptical in face view. Their dimensions are 136.4 nm by 91.0 nm by 81.8 nm high. In side view, the scale is seen to have a thick lower half and a thin upper half. This scale type is very different from those of previously reported Amphidinium species (HG114 and HG115). The molecular tree indicated that A. cupulatisquama and the two other strains of body scale-bearing Amphidinium are distantly related within the Amphidinium clade.

Symbiotic dinoflagellates of the species Amphidinium are expected to be pharmaceutically useful microalgae because they produce antitumor macrolides. A microalgae production system with a large number of cells at a high density has been developed for the efficient production of macrolide compounds. In the present study, the effects of culture conditions on the cellular growth rate of dinoflagellates were investigated to determine the optimum culture conditions for obtaining high yields of microalgae. Amphidinium species was cultured under conditions with six temperature levels (21–35°C), six levels of photosynthetic photon flux density (15–70 μmol photons m−2 s−1), three levels of CO2 concentration (0.02–0.1%), and three levels of O2 concentration (0.2–21%). The number of cells cultured in a certain volume of solution was monitored microscopically and the cellular growth rate was expressed as the specific growth rate. The maximum specific growth rate was 0.022 h−1 at a temperature of 26°C and O2 concentration of 5%, and the specific growth rate was saturated at a CO2 concentration of 0.05%, a photosynthetic photon flux density of 35 μmol photons m−2 s−1 and a photoperiod of 12 h day−1 upon increasing each environmental parameter. The results demonstrate that Amphidinium species can multiply efficiently under conditions of relatively low light intensity and low O2 concentration.

Identification of novel dinoflagellate taxa through molecular analysis is hindered by lack of well-defined basal lineages. To address this issue, we attempted to reassess the phylogenetic status of Oxyrrhis marina Dujard. as well as other potentially basal taxa. The analysis was based on two newly established premises: (1) editing density of mitochondrial cob and cox1 mRNA increases from basal to later diverging lineages; (2) nuclear-encoded mRNA in dinoflagellates is trans-spliced to receive a 22 bp spliced leader (SL) at the 5′-end. We analyzed these two genetic traits in O. marina, Noctiluca scintillans (Macartney) Kof. et Swezy, Heterocapsa triquetra (Ehrenb.) F. Stein, H. rotundata (Lohmann) Ge. Hansen, Amphidinium carterae Hulburt, and A. operculatum Clap. et J. Lachm. Surprisingly, no editing was detected in cob and cox1 mRNAs in these lineages, except for a small number of editing events in Amphidinium. However, nuclear-encoded mRNAs in these species contained the SL sequence at the 5′-end, indicative of SL RNA trans-splicing. These findings, together with the recent cob-cox1-18S rRNA three-gene phylogeny, suggest the following: (1) O. marina is a basal dinoflagellate; (2) Heterocapsa, Amphidinium, and Noctiluca likely are also early diverging lineages of dinoflagellates, and the position of Heterocapsa is inconsistent with literature and needs further investigation; and (3) the presence of the 22 bp SL and mitochondrial (mt) mRNA editing can be considered a landmark of dinoflagellate splits.

Since its description in 1917, the taxonomy of Katodinium glaucum, a cosmopolitan heterotrophic marine dinoflagellate, has been in a state of flux. We added to this by arguing that Kat. glaucum is synonymous with both Amphidinium extensum and Gymnodinium vestifici. We presented a brief historical account of its shifting positions in Katodinium and Gyrodinium and a comparison with Gym. vestifici and A. extensum. The conclusions from our experimental work on a Kat. glaucum culture were based on both pheno- and genotypic characters obtained by light and electron microscopy and DNA sequencing. A nuclear-encoded large-subunit rDNA-based phylogeny revealed that Kat. glaucum and species of Torodinium are closely related and form a highly supported monophyletic lineage. The ultrastructural examination showed the apical structure complex of Kat. glaucum to comprise three rows of amphiesmal vesicles located beneath the cap-like projection on the anterior part of the cell. Torodinium, in comparison, possessed a counterclockwise spiral apical structure complex positioned on top of its bill-like apical projection. Species of both genera were longitudinally striated. The type species of Katodinium, Kat. nieuportense, was illustrated without any apical structures and without longitudinal striations, and because both morphological and molecular analysis also rejected relationship to Amphidinium, Gymnodinium and Gyrodinium, we proposed to erect a new genus for this species, Kapelodinium gen. nov. As the species name vestifici had priority over glaucum, the new name for the species was Kapelodinium vestifici comb. nov. To accommodate for the distinct difference in morphology of the apical structure complex in Torodinium and Kapelodinium, we further proposed two new monotypic families, Torodiniaceae fam. nov. and Kapelodiniaceae fam. nov. of the order Torodiniales ord. nov., which in the molecular trees was not closely related to other dinoflagellates. The ultrastructural study of Kap. vestifici gen. & comb. nov. also revealed a number of characteristic features, such as a novel lip-like suture within the sulcus, cross-striated filaments beneath the apical projection and atypical nuclear chambers with pores towards both the nucleoplasm and the cytoplasm.

Three hundred and sixty-nine macroalgal and non-algal samples were collected from six coastal sites around the island of Hawai‘i on a biweekly basis over a 1-year period to ascertain (1) the presence of potentially toxigenic benthic dinoflagellates, and (2) substrate and environmental preferences of the dinoflagellates. Twenty-six genera/species of dinoflagellates were encountered including the (potentially) toxigenic species Amphidinium sp., Coolia monotis, Gambierdiscus sp., Ostreopsis ovata, Prorocentrum concavum, Prorocentrum hoffmannianum, Prorocentrum lima, and Prorocentrum mexicanum. Twenty of the species are being reported for the first time as present in Hawaiian coastal waters. There was some evidence of macroalgal host preference (e.g., C. monotis on Tolypiocladia glomerulata), although host morphology preferences was greater (e.g., Gambierdiscus sp., and P. lima on filamentous turfs; O. sp. 1 on sheet-like macroblades). While some dinoflagellate groups were significantly correlated with nutrient concentrations (e.g., total dinoflagellates with nitrate + nitrite and phosphate concentrations), others were not (e.g., Gambierdiscus sp., Prorocentrum emarginatum, P. lima, and Sinophysis microcephalus). The presence of several potentially toxigenic dinoflagellate species in Hawai‘i merits future study on possible impacts of these dinoflagellates on coastalfood websand humanhealth. # 2007 Elsevier B.V. All rights reserved.

Two major groups of dinoflagellates can be distinguished based on their cell coverings. Thecate (armored) dinoflagellates with large amphiesmal vesicles filled with cellulosic material, and the athecate (unarmored or “naked”) dinoflagellates that contain hundreds of alveoli lacking cellulosic material (Morrill and Loeblich 1983). The naked dinoflagellates are usually fragile and delicate, the cells easily lyse during the observation of live samples, lyse due to the fixation, or the fixed cells are too distorted for proper identification. Particularly, gymnodinioid dinoflagellates are notoriously difficult to preserve. The chemical fixatives produce misshapen cells, swollen membranes, and clumping of specimens (Kofoid and Swezy 1921). However, the separation between armored and unarmored species is not clear cut and some gymnodinioid dinoflagellates are characterized by a thick cell covering. This is the case of the genus Balechina Loeblich et A.R. Loeblich, which has been placed either in the order Kolkwitziellales (Taylor 1987) or Ptychodiscales (Fensome et al. 1993). Kofoid and Swezy (1921) published a monograph of the unarmored dinoflagellates known at that time. They proposed the subgenus Pachydinium Kofoid et Swezy for gymnodinioid species with a thick cell covering. They included three new species lacking surface longitudinal ridges (Gymnodinium pachydermatum Kofoid et Swezy, G. dogielii Kofoid et Swezy, and G. amphora Kofoid et Swezy), and species with a surface covered by longitudinal ridges (Gymnodinium coeruleum Dogiel, and the new species G. canus Kofoid et Swezy, G. costatum Kofoid et Swezy, and G. lira Kofoid et Swezy). Despite the large sizes and thick cell covering of these species, only G. coeruleum has sporadically been reported in the literature, while the records of the other species are very rarely reported or only known from the original descriptions (Dogiel 1906, Kofoid and Swezy 1921, Wood 1968). This may be due to the fact that G. coeruleum is characterized by a striking blue or purple coloration, which is relatively rare in nature, in particular in microbial species. With no known observations, Loeblich and Loeblich (1968) proposed that the subgenus Pachydinium should be raised at the genus rank. They proposed the new name Balechina because Pachydinium Kofoid et Swezy 1921 was a junior homonym of the thecate Pachydinium Pavillard 1915. This proposal was followed only by Taylor (1976), who transferred G. coeruleum into Balechina, and proposed a third species Balechina marianiae F.J.R. Taylor (see Appendix S1 in the Supporting Information for a taxonomic, nomenclatural and biogeographical account). The genus Balechina was further used in the literature (Lessard and Swift 1986, Taylor 1987, Fensome et al. 1993, Steidinger and Tangen 1997), while other authors considered Balechina as a synonym of Gymnodinium F. Stein (Sournia 1986, Balech 1988). In the last 15 years our knowledge of the unarmored dinoflagellates has increased with the advances of molecular phylogeny, initially based mostly on photosynthetic cultivable species (Daugbjerg et al. 2000), abundant heterotrophic coastal species (Hansen and Daugbjerg 2004, Takano and Horiguchi 2004) and later, on other less abundant heterotrophic species (Gomez et al. 2009). However, little is known about the unarmored dinoflagellates with a thick cell covering that reach larger sizes (>200 µm long, i.e., Gymnodinium cucumis F. Schutt), or highly distinctive species with striking blue or purple pigmentation (i.e., Balechina coerulea (Dogiel) F.J.R. Taylor). To the best of our knowledge, the type species of Balechina, B. pachydermata (Kofoid et Swezy) Loeblich et A.R. Loeblich, remains only known from the original description (Kofoid and Swezy 1921) and Wood (1968). This study was based on observations of live material from several locations of the Mediterranean Sea (Marseille, Banyuls sur Mer, Villefranche sur Mer, Valencia), the South Atlantic Ocean on the coast of Brazil (Sao Sebastiao Channel and off Ubatuba), and the North Pacific Ocean on the coast of Japan (Kure, Hiroshima Prefecture). We provided the first micrographs of the species B. pachydermata, G. cucumis, G. dogielii, G. amphora and Amphidinium vasculum, and scanning electron microscopy pictures of the species B. coerulea, B. pachydermata, and G. lira, including their apical grooves. We reported for the first time the phenomenon of the sudden contraction of B. pachydermata, and its high intraspecific variability, mainly in the shape of the episome. We propose the species A. vasculum Kofoid et Swezy, G. amphora, G. dogielii and G. gracile sensu Kofoid and Swezy as synonyms of B. pachydermata. We also illustrated the phenomenon of autotomy in an unarmored dinoflagellate based on our observations of B. coerulea. We propose the species G. canus and G. costatum as synonyms of B. coerulea. We provided the first molecular data (SSU rRNA gene sequences) of the species B. pachydermata, B. coerulea, Gymnodinium amphora, G. cucumis and G. lira. We propose an emended description of the genus Balechina, a new genus for the species B. coerulea, G. cucumis, G. lira, and a tentative undescribed species with intermediate characteristics between B. coerulea and G. lira.

We report on a dense bloom (~1.80 × 10(5) cells mL(−1)) of the marine dinoflagellate species Amphidinium carterae (Genotype 2) in a shallow, small intermittently open coastal lagoon in south eastern Australia. This bloom co-occurred with the deaths of >300 individuals of three different species of fish. The opening of the lagoon to the ocean, as well as localized high nutrient levels, preceded the observations of very high cell numbers. A. carterae is usually benthic and sediment-dwelling, but temporarily became abundant throughout the water column in this shallow (

The examination of macrophyte, water and sediment samples, collected at depths less than 1.5 m from 50 different sites along the North Aegean coasts, has revealed, for the first time in Greek coastal waters, the presence of two Ostreopsis species (O. ovata and O. cf. siamensis) and Coolia monotis in the majority of the sampling sites (94% and 100%, respectively). Other epiphytic dinoflagellates of thegenera Prorocentrumand Amphidinium and diatoms were accompanying species in this epiphytic community. Morphometric features, plate formula and thecal ornamentation were used for species identification. O. ovata cells were smaller in dorsoventral (DV) diameter and width (W) (26.18‐61.88 mm and 13.09‐47.60 mm, respectively) in comparison with O. cf. siamensis (35.70‐65.45 mm and 23.80‐49.98 mm, respectively). In contrast, the anterioposterior (AP) diameter of O. cf. siamensis was smaller (14.28‐26.18 mm) resulting in DV/AP � 3, whereas the above ratio for O. ovata was less than 2 (AP ranging between 14.28‐35.70 mm). Moreover, the theca of O. ovata cells was ornamented with scattered pores, which fluctuated in a wider range (0.07‐0.32 mm) than those of O. cf. siamensis (0.23‐0.29 mm). Coolia monotis cells were almost round with average DV diameter 26.88 mm, AP 25.66 mm and width 26.76 mm. Small and large cells were recorded in both field and culture populations of Ostreopsis spp. and C. monotis, while hyaline cysts were observed for O. ovata. The presence of O. ovata and O. cf. siamensis exhibited a clear seasonal pattern dominating (maximum abundance up to 4.05 � 10 5 cells gr � 1 fwm) the period from midsummer

The occurrence and toxicity of Amphidinium carterae Hulburt is hereby reported for the first time from the North Arabian Sea on the coast of Pakistan. The concentrations of 1.2 × 10 4 cells ml −1 were found in intertidal pools that were also inhabited by the brown macroalga Sargassum wightii. Both wild and cultured A. carterae cells were tested for ciguatera toxicity through exposure to brine shrimp nauplii ( Artemia salina ) and albino mice. Although the brine shrimp did not appear to be affected mortalities in mice ranged between 13 and 16% at doses of 7.2 × 10 4 and 2.5 × 10 5 cells ml −1 , respectively. When mice were affected pharmacological effects such as muscle contraction in lower back area, increased respiration, immobility and paralysis in hind limbs were observed for 2 h. These effects appeared to be reversible and gradually disappeared within 24 h.

Se presenta una lista taxonómica con anotaciones de aproximadamente 605 especies y taxones infraespecíficos de dinoflagelados principalmente de vida libre comprendidos en 91 géneros que han sido reportados para el Pacífico Mexicano en la literatura publicada, algunas tesis, reportes no publicados y datos originales. Las referencias de la literatura entre 1878 y 2005 (en total, 311) están incluidas en la bibliografía, y en las notas taxonómicas concernientes a los taxa proporcionados, donde se consideró apropiado. La presencia de ilustraciones está dividida en tres categorías (dibujos, fotografías en microscopio óptico, y fotografías en microscopio electrónico de barrido) y se indica la distribución de los taxa en el Pacífico Mexicano. La nomenclatura y 193 especies y taxa infraespecíficos. Cuatro nuevas combinaciones: Histioneis pieltainii (B. F. Osorio) Okolodkov comb. nov., Latifascia subantarctica (Balech) Okolodkov comb. nov., Peridiniella globosa (P. A. Dang.) Okolodkov comb. nov. y Protoperidinium curtipes (Jörg.) Balech f. asymmetricum (Matzen.) Okolodkov comb. nov. Veintinueve nomina nuda son revelados. Los géneros mas importantes son Protoperidinium (111 especies), Ceratium (63), Dinophysis (41), Gonyaulax (25), Oxytoxum (22), Gymnodinium (22), Prorocentrum (21), Alexandrium (17), Ornithocercus (12) y Amphidinium (12). Para conocer la diversidad real de las especies de dinoflagelados en el Pacífico Mexicano, se necesitan hacer más estudios sobre las especies atecados de los Gymnodiniales, los dinoflagelados bentónicos y epifíticos, el ‘‘grupo Diplopsalis’’, el género Protoperidinium y los géneros recientemente descritos de la familia Podolampadaceae.

Amphidinium is a genus of naked dinoflagellates, characterized by a small epicone and a larger hypocone. Presently about 120 species from both freshwater and marine biotopes have been described. The genus has long been known to be poly-phyletic, however, and the recent rediscovery of the type species has now allowed for more precise definition of the genus. The new circumscription of the genus leaves the freshwater species without a generic name. A new generic name, Prosoaulax gen. nov., is therefore proposed for the first described freshwater species, Amphidinium lacustre Stein (1883), and related species. Based on ultrastructural features, notably the very unusual type of eyespot, Prosoaulax is considered to be related to a recently recognized, but poorly understood group of dinoflagellates, comprising the marine species Gymnodinium simplex, G. natalense, Polarella antarctica, and some symbionts of marine invertebrates, G. bei and G. linucheae. It also includes the symbionts of corals (‘zooxant...

Amphidinols, antifungal and hemolytic compounds, were isolated from the marine dinoflagellates Amphidinium spp. Their structures were characterized by a conjugated triene, two ether rings, and polyhydroxy groups. Five new amphidinol analogs, amphidinols 9, 10, 11, 12 and 13 were isolated together with amphidinol 2 (AM2), and amphidinol 4 (AM4) from the dinoflagellate, Amphidinium carterae collected in New Zealand. Their structures were elucidated by detailed analyses of MS and 2D NMR spectra. Amphidinol 9 was a regioisomer of AM3, and amphidinol 10 was dinorAM4. Amphidinol 11, 12 and 13 possess a sulfate ester at C1 of AM2, AM4, and AM9, respectively. Attachment of the sulfate ester markedly reduced their antifungal and hemolytic activities.

SUMMARY The recent emendation of Amphidinium (Dinophyc-eae), which now only consists of species with minute left-deflected epicone, has left more than 100 species without a clear generic affiliation. In the present study, a strain identified as one of the species with a divergent epicone type, Amphidinium britannicum (Herdman) Lebour, and six strains resembling A. britannicum but smaller in size were examined by light, scanning and transmission electron microscopy and by sequence analyses of nuclear-encoded partial large subunit ribosomal DNA to establish their phylog-eny. Amphidinium britannicum was not closely related to other genera included in the molecular phylogenetic analyses, but formed a highly supported clade in Bayesian analysis together with the six small-sized strains. The six strains also formed a highly supported clade, consisting of two closely related, albeit distinct, clades. Light and scanning electron microscopy did not reveal significant differences between the vegetative motile cells; however, cells about to undergo mitosis developed longitudinal grooves on the hypocone in one of the clades but not in the other. Both clades differed substantially from A. britannicum in partial large subunit ribosomal DNA as well as in size and shape. Based on morphological similarity and partial large subunit ribosomal DNA evidence, we erect the new genus, Togula gen. nov. with the emended type species Togula britannica (Herdman) comb. nov. Based on differences in division pattern and partial large subunit ribosomal DNA gene divergence we further describe the species Togula compacta (Herdman) comb. nov. and Togula jolla sp. nov.

Amphidinium operculatum Claparede et Lachmann, the type species of the dinoflagellate genus Amphidinium, has long had an uncertain identity. It has been considered to be either difficult to distinguish from other similar species or a morphologically variable species itself. This has led to the hypothesis that A. operculatum represents a “species complex.” Recently, the problem of distinguishing A. operculatum from similar species has become particularly acute, because several morphologically similar species have been found to produce bioactive compounds of potential interest to the pharmaceutical industry. In this study, we cultured and examined existing cultures of several species of Amphidinium, most of which have been previously identified as A. operculatum or as species considered by some to be synonyms or varieties of A. operculatum. Thirty strains were examined using comparative LM, SEM, and partial large subunit (LSU) rDNA sequence data. Through morphological and molecular phylogenetic analyses, six distinct species were identified, including Amphidinium trulla sp. nov. and Amphidinium gibbosum comb. nov. Amphidinium operculatum was redescribed based on four cultures. Genetic variability within the examined Amphidinium species varied greatly. There was little difference among strains in partial LSU rDNA for most species, but strains of A. carterae and A. massartii Biencheler differed by as much as 4%. In both A. carterae and A. massartii, three distinct genotypes based on partial LSU rDNA were found, but no morphological differences among strains could be observed using LM or SEM. In the case of A. carterae, no biogeographically related molecular differences were found.

Members of Amphidinium Claparede and Lachmann constitute a major part of sand-dwelling benthic dinoflagellates worldwide. The genus is traditionally defined by its small epicone size, not exceeding one third of the total cell length. It has long been suspected that this functional definition does not reflect phylogeny, yet the problem of identifying the type species A. operculatum and closely related species has until now hindered attempts to redefine the genus. In this study 12 Amphidinium species were examined using phylogenetic analyses based on nuclear-encoded, partial, large subunit (LSU) rDNA, with a further six Amphidinium species being included in a morphological cladistic analysis. The species selected represented taxa with a range of morphological dissimilar epicone forms. Both cladistic analysis and analyses based on partial LSU rDNA revealed that Amphidinium species with minute left-deflected epicones formed a monophyletic clade that included the type species. Amphidinium species with other epicone types were found to be unrelated to this clade. The type species A. operculatum was identified based on general cell shape and size, position of a dark organelle previously defined as a stigma, and origin of the sulcus. The description of A. elegans by Grell and Wohlfarth-Bottermann was found to be identical to it. A species fitting the original description of A. operculatum was cultured and included in the analyses. Based on cladistic and molecular analyses, it grouped together with all other species with minute left-deflected epicones, and this group constitutes the true genus Amphidinium sensu stricto. An emendation of the genus definition is presented.

A novel type of body scale has been found in two strains of unidentified species of the dinoflagellate genus Amphidinium. The body scales are distributed over the entire outer surface of the plasma membrane of the cells, and are embedded in an amorphous material. No scales are present on the flagellar membrane. The body scales are oval, uniform and simple, and lack a basal plate; they are similar in shape in the two species, but differ in size. Scale formation takes place within the Golgi cisternae. The mature scales are then transported to the vicinity of the flagellar canal in Golgi vesicles in the cytoplasm, where they are extruded out of the cell. These two species are morphologically indistinguishable from Amphidinium carterae except by the presence of scales, and this distinction is supported by base pair differences in the small subunit ribosomal RNA (SSU rRNA) gene (28–29 base alterations). We consider the two strains to be different species on the basis of differences in the size of the ...

Seventeen species of the mainly sand-dwelling genus Amphidinium are reported from south-eastern Australian waters. Three new species are described: A. boggayum, A. mootonorum and A. yuroogurrum. Additional features from previously described species are presented, including non-motile cell stages. Nine of these species are new records for Australia; however, these species are probably widespread and common. Two potentially ichthyotoxic species of Amphidinium were commonly found: A. carterae and A. cf. operculatum. A table of morphological characters is compiled as a step towards recognizing new characters with which to group species of this presently artificially defined unarmoured dinoflagellate genus.

Light microscopy and scanning electron microscopy were used to study Amphidinium glabrum sp. nov., a new dinoflagellate species from tidal sand flats in the North German Wadden Sea and from the surface water and the lowermost layer of first-year ice in the European Arctic. Cells are strongly flattened dorso-ventrally, with nearly parallel sides in ventral view. The distal region of the strongly displaced cingulum is not connected with the sulcus. There is no surface striation. The sulcus extends from the antapex, where it forms a large excavation, to the apex and further onto the dorsal side of the epicone. The new species is compared with two morphologically similar dinoflagellates: A. scissum Kofoid & Swezy and A. scissoides Lebour. Characters that distinguish the three taxa are: cell shape and size, shape of epicone and hypocone, cell surface, cingulum displacement, distal region of the cingulum and antapical notch.

Summary Genetic transformation of two dinoflagellates (Amphidinium sp., Symbiodinium microadriaticum) was achieved using plasmid constructs containing the neomycin phosphotransferase gene (nptII) fused to the Agrobacterium nos promoter, or the hygromycin B phosphotransferase gene (hpt) fused to the bidirectional Agrobacterium p1′2′ promoter. Gene transfer into intact (walled) dinoflagellate cells was achieved by agitation in the presence of silicon carbide (SiCa) whiskers. Transformation rates of 5–24 transformants per 107 cells were obtained. Southern hybridization of transformants revealed stable integration of multiple copies of the constructs. Activity of integrated copies of the β-glucoronidase (GUS) reporter gene coupled to the cauliflower mosaic virus 35S promoter or the p1′2′ promoter was confirmed both histochemically and fluorometrically. This is the first report of successful application of heterologous and widely used promoter and reporter genes in microalgae, and is the first demonstration of transformation of a dinoflagellate. There appear to be no substantial barriers to transformation of Amphidinium and Symbiodinium, which must now be considered as the first of the dinoflagellate genera accessible to genetic manipulation.

The major light-harvesting complex of Amphidinium (A.) carterae, chlorophyll-a-chlorophyll-c 2-peridinin-protein complex (acpPC), was studied using ultrafast pump-probe spectroscopy at low temperature (60 K). An efficient peridinin-chlorophyll-a energy transfer was observed. The stimulated emission signal monitored in the near-infrared spectral region was stronger when redder part of peridinin pool was excited, indicating that these peridinins have the S1/ICT (intramolecular charge-transfer) state with significant charge-transfer character. This may lead to enhanced energy transfer efficiency from "red" peridinins to chlorophyll-a. Contrary to the water-soluble antenna of A. carterae, peridinin-chlorophyll-a protein, the energy transfer rates in acpPC were slower under low-temperature conditions. This fact underscores the influence of the protein environment on the excited-state dynamics of pigments and/or the specificity of organization of the two pigment-protein complexes.

A single-cell isolate of Amphidinium from the northern Caribbean Sea was identified as A. operculatum Clap. et Lack. var. nov. gibbosum Maranda et Shimizu based on a morphological and ultrastructural study. This free-swimming dinoflagellate, which was found to produce potent antitumor metabolites, is elongate and asymmetrical. It is compared with two close relatives A. klebsii Kofoid et Swezy and A. carterae Hulburt. Amphidinium operculatum var. gibbosum can be distinguished from A. klebsii on the basis of shape while it differs from A. carterae on the basis of size, shape, cell surface, and chloroplast arrangement. The shape and size of the nucleus and the presence of mucocysts also differentiate the gibbosum variety from its two relatives. The architecture of the pusule may ultimately provide the best ultrastructural discriminating character. Otherwise, the general ultra-structure of the Caribbean isolate is typical of many unarmored photo synthetic dinoflagellates with amphiesma, condensed chromosomes, trichocysts, mitochondria with tubular cristae, and trilamellar chloroplasts.

SUMMARY A new sand-dwelling dinoflagellate is described from Sesoko Beach, Okinawa Island, subtropical Japan and its micromorphology is studied by means of light and electron microscopy. The cell consists of a small epitheca and a large hypothecs superficially resembling members of the unarmored genus Amphidinium. The cell is dorso-ventrally flattened and possesses a single chloroplast with a large conspicuous pyrenoid. Transmission electron microscopy revealed that the dinoflagellate possesses typical dinoflagellate cellular organization. Scanning electron microscopy demonstrated that the organism is thecate and the thecal plate arrangement is Po, 4′, 1a, 7″, 5c, 4s, 6″′, 2″″. Most of the characteristics suggest gonyaulacalean affinity of the new species. These are the presence of ventral pore, lack of canal plate, direct contact between the sulcal anterior plate and the flagellar pore, possession of six postcingular plates and asymmetrical arrangement of the antapical plates. Affinity to existing families of the order Gonyaulacales has not been determined. Based on the unique cell shape, thecal plate arrangement and the presence of ventral pore, a new genus, Amphidiniella, is established for this organism and the species is named A. sedentaria Horiguchi gen. et sp. nov.

The small-subunit ribosomal RNA genes (SSU rDNA) from the four symbiotic dinoflagellates, Symbiodinium corculorum Trench isolated from the bivalve mollusc Corculum cardissa (from Belau, Western Caroline Is.), S. meandrinae Trench, from the scleractinian coral Meandrina meandrites (from famaica, W.I.), Gloeodinium viscum Banaszak et al. from the hydrocoral Millepora dichotoma (from the Gulf of Aqaba), and Amphidinium belauense Trench from the acoel flatworm Haplodiscus sp. (from Belau) have been amplified by the polymerase chain reaction, cloned, and sequenced. Following alignment of these complete sequences to homologous sequences from six other dinoflagellates, eight api-complexans, six ciliates, six chromophytes and oomycetes, three ascomycetes, two rhodophytes, two chlorophytes, and two myxomycetes (with Physarum polycephalum as the outgroup), phylogenetic reconstruction was conducted using Fitch and Margoliash distance, DNA maximum likelihood, and Wagner parsimony methods, with bootstrap resampling. All methods generated trees with similar topologies. The inferred “across Kingdom” phylogeny reemphasizes previous reports that show that the dinoflagellates, the apicomplexans, and the ciliates share a common ancestry and that the dinoflagellates are distantly related to the chromophyte-oomycete lineage. The evidence supports the concept of a polyphyletic origin of dinoflagellate-invertebrate symbioses, as symbiotic dinoflagellates represent seven genera in at least four orders. The three symbiotic species, S. corculorum, S. meandrinae, and S. pilosum, consistent with their morphological and biochemical similarities, cluster most closely. Symbiodinium pulchrorum Trench, the symbiontfrom the Hawaiian sea anemone Aiptasia pulchella, is more distantly related to them. Gloeodinium viscum is not closely related to the Symbiodinium species. Amphidinium carterae (free-living) and A. belauense (symbiotic) also appear to be distantly related to Symbiodinium. Some symbionts (e.g. S. corculorum, S. pilosum) from distant geographic locations (the Indo-Pacific and Caribbean, respectively) were found to be very closely related, whereas S. pulchrorum and S. corculorum from the Pacific were found to be distantly related. Analyses of 10 additional symbiotic and nonsymbiotic dinoflagellates, using partial SSU rDNA sequences to generate a tentative dinoflagellate phylogeny, indicate that members of the genus Symbiodinium cluster with most of the other (free-living) dinoflagellates in the genus Gymnodinium. The genus Amphidinium, as represented by A. carterae and A. belauense, appear to be distantly related to the other members of the Gymnodiniaceae. This analysis, combined with morphological and biochemical data, indicates that the symbionts S. pulchrorum (from Aiptasia pulchella) and S. bermudense Trench (from Aiptasia tagetes) from the Indo-Pacific and Caribbean, respectively, are very closely related but are not identical.

The cold-water, photosynthetic dinoflagellate Amphidinium cryophilum Wedemayer, Wilcox & Graham feeds phagotrophically on other dinoflagellate species. Food is ingested through a feeding tube, termed here the “phagopod,” which extends from the antapex. The peduncle of this organism plays no observable role in the feeding process. The phagopod is essentially a hollow cylinder composed electron-opaque material that is possibly deposited on a membrane. No Amphidinium cytoplasmic components, including microtubules or other cytoskeletal elements, were observed in the phagopod. Prefeeding cells aggregate, in small clumps near prey organisms with their phagopods extended. Eventually some cells commence feeding, first inserting the phagopod through the prey cell-covering and then slowly, over a period of 10 min or more, drawing cytoplasm through the phagopod and into a nascent food vacuole. Both light and electron microscopy suggest that one or more prey cell amphiesmal membranes remain intact during the feeding process. Upon completion of feeding, the Amphidinium cell swims off with a prominent food vacuole in the hypocone, leaving at least part of the phagopod attached to the prey cell. Phagotrophy in A. cryophilum seems to vary with light intensity. At low light intensities, cells feed phagotrophically and are nearly colorless, whereas at high light levels they feed much less frequently, if at all, and are brightly pigmented.

Gymnodinium acidotum Nygaard is a freshwater dinoflagellate that is known to harbor a cryptomonad endosymbiont whose chloroplasls give the organism an overall blue-green color. The ultrastructure of G. acidotum was examined with particular attention being given to the three dimensional nature of the flagellar apparatus. The fiagellar apparatus is composed of two functional basal bodies that are slightly offset and lie at an angle of approximately 90° to one another. As in other dinoflagellates the transverse basal body is associated with a striated, fibrous root that extends from the proximal end of the basal body to the transverse flagellar opening. At least one microtubular root extends from the proximal end of the transverse basal body, and a multi-membered longitudinal microtubular root is associated with the longitudinal basal body. The most striking feature of the flagellar apparatus of G. acidotum is the large fibrous connective that extends from the region of the proximal ends of the basal bodies to the cingulum on the dorsal side of the cell. A similar structure has been reported from only one other dinoflagellate, Amphidinium cryophilum Wedemayer, Wilcox, and Graham. The presence of this structure as well as similarities in external morphology suggest thai these two species may be more closely related to each other than either is to other gymnodinioid taxa. The taxonomic importance of dinoflagellate flagellar apparatus components is discussed.

The flagellar apparatus of the marine dinoflagellate Amphidinium rhynchocephalum Anissimowa was examined using the techniques of rapid freezing/freeze substitution and serial thin section three dimensional reconstruction. The flagellar apparatus is composed of two basal bodies that are offset from one another and lie at an angle of approximately 150° The transverse basal body is associated with two individual microtubules that extend from the proximal end of the basal body toward the flagellar opening. One of these microtubules is closely appressed to a striated fibrous root that also extends from the proximal base of the transverse basal body. The longitudinal basal body is associated with a nine member microtubular root that extends from the proximal end of the basal body toward the posterior of the cell. The longitudinal microtubular root and the transverse striated fiber are connected by a striated connective fiber. In addition to the microtubules associated with the transverse and longitudinal basal bodies, a group of microtubules originates adjacent to one of the transverse flagellar roots and extends into the cytoplasm. Vesicular channels extend from the flagellar openings to the region of the basal bodies where they expand to encompass the various connective structures of the flagellar apparatus. The possible function and evolutionary importance of these structures is discussed.