Does dinocyst wall composition really reflect trophic affinity? New evidence from ATR micro-FTIR spectroscopy measurements.

Attenuated total reflection (ATR) microscope Fourier transform infrared (micro-FTIR) spectroscopy was used to investigate the dinosporin composition in the walls of modern, organic-walled dinoflagellate resting cysts (dinocysts). Variable cyst wall compositions were observed, which led to the erection of four spectrochemical groups, some with striking similarities to other resistant biomacromolecules such as sporopollenin and algaenan. Furthermore, possible proxies derivable from the spectrochemical composition of modern and fossil dinocysts were discussed. The color of the dinocyst walls was reflected in the spectral data. When comparing that color with a standard and the results of a series of bleaching experiments with oxidative agents, eumelanin was assigned as a likely pigment contributing to the observed color. Following this assignment, the role of eumelanin as an ultraviolet sunscreen in colored dinocysts was hypothesized, and its implications on the autofluorescence and morphological preservation of dinocysts were further discussed. Unlike what had previously been assumed, it was shown that micro-FTIR data from dinocysts cannot be used to unambiguously infer trophic affinities of their associated cells. Finally, using methods with high spatial resolutions (synchrotron transmission micro-FTIR and optical photothermal infrared spectroscopy), it was shown that dinocyst wall layers are chemically homogenous at the probed scales. This study fills a large knowledge gap in our understanding of the chemical nature of dinocyst walls and has nuanced certain assumptions and interpretations made in the past.

Unraveling the Gonyaulax baltica Species Complex: Cyst-theca Relationship of Impagidinium variaseptum, Spiniferites pseudodelicatus sp. nov. and S. ristingensis (Gonyaulacaceae, Dinophyceae), With Descriptions of Gonyaulax bohaiensis sp. nov, G. amoyensis sp. nov. and G. portimonensis sp. nov.

The taxonomy of the extant dinoflagellate genus Gonyaulax is challenging since its thecate morphology is rather conservative. In contrast, cysts of Gonyaulax are varied in morphology and have been related with the fossil-based genera Spiniferites and Impagidinium. To better understand the systematics of Gonyaulax species, we performed germination experiments on cysts that can be identified as S. ristingensis, an unidentified Spiniferites with petaloid processes here described as Spiniferites pseudodelicatus sp. nov. and Impagidinium variaseptum from Chinese and Portuguese waters. Despite marked differences in cyst morphology, motile cells of S. pseudodelicatus and I. variaseptum are indistinguishable from Gonyaulax baltica. Motile cells hatched from S. ristingensis are morphologically similar to G. baltica as well but differ in the presence of one pronounced antapical spine. Three new species, Gonyaulax amoyensis (cyst equivalent S. pseudodelicatus), Gonyaulax bohaiensis (cyst equivalent I. variaseptum), and Gonyaulax portimonensis (cyst equivalent S. ristingensis), were erected. In addition, a new ribotype (B) of G. baltica was reported from South Korea and a bloom of G. baltica ribotype B is reported from New Zealand. Molecular phylogeny based on LSU and SSU rRNA gene sequences revealed that Gonyaulax species with minute or short antapical spines formed a well-resolved clade, whereas species with two pronounced antapical spines or lack of antapical spines formed the sister clade. Six strains of four above species were examined for yessotoxin production by liquid chromatography coupled with tandem mass spectrometry, and very low concentrations of yessotoxin were detected for one G. bohaiensis strain.

Monitoring the Emergence of Algal Toxins in Shellfish: First Report on Detection of Brevetoxins in French Mediterranean Mussels.

In France, four groups of lipophilic toxins are currently regulated: okadaic acid/dinophysistoxins, pectenotoxins, yessotoxins and azaspiracids. However, many other families of toxins exist, which can be emerging toxins. Emerging toxins include both toxins recently detected in a specific area of France but not regulated yet (e.g., cyclic imines, ovatoxins) or toxins only detected outside of France (e.g., brevetoxins). To anticipate the introduction to France of these emerging toxins, a monitoring program called EMERGTOX was set up along the French coasts in 2018. The single-laboratory validation of this approach was performed according to the NF V03-110 guidelines by building an accuracy profile. Our specific, reliable and sensitive approach allowed us to detect brevetoxins (BTX-2 and/or BTX-3) in addition to the lipophilic toxins already regulated in France. Brevetoxins were detected for the first time in French Mediterranean mussels (Diana Lagoon, Corsica) in autumn 2018, and regularly every year since during the same seasons (autumn, winter). The maximum content found was 345 µg (BTX-2 + BTX-3)/kg in mussel digestive glands in November 2020. None were detected in oysters sampled at the same site. In addition, a retroactive analysis of preserved mussels demonstrated the presence of BTX-3 in mussels from the same site sampled in November 2015. The detection of BTX could be related to the presence in situ at the same period of four Karenia species and two raphidophytes, which all could be potential producers of these toxins. Further investigations are necessary to understand the origin of these toxins.

Morphology, ultrastructure, and molecular phylogeny of Wangodinium sinense gen. et sp. nov. (Gymnodiniales, Dinophyceae) and revisiting of Gymnodinium dorsalisulcum and Gymnodinium impudicum.

The genus Gymnodinium includes many morphologically similar species, but molecular phylogenies show that it is polyphyletic. Eight strains of Gymnodinium impudicum, Gymnodinium dorsalisulcum and a novel Gymnodinium-like species from Chinese and Malaysian waters and the Mediterranean Sea were established. All of these strains were examined with light microscopy, scanning electron microscopy and transmission electron microscopy. SSU, LSU and internal transcribed spacers rDNA sequences were obtained. A new genus, Wangodinium, was erected to incorporate strains with a loop-shaped apical structure complex (ASC) comprising two rows of amphiesmal vesicles, here referred to as a new type of ASC. The chloroplasts of Wangodinium sinense are enveloped by two membranes. Pigment analysis shows that peridinin is the main accessory pigment in W. sinense. Wangodinium differs from other genera mainly in its unique ASC, and additionally differs from Gymnodinium in the absence of nuclear chambers, and from Lepidodinium in the absence of Chl b and nuclear chambers. New morphological information was provided for G. dorsalisulcum and G. impudicum, e.g., a short sulcal intrusion in G. dorsalisulcum; nuclear chambers in G. impudicum and G. dorsalisulcum; and a chloroplast enveloped by two membranes in G. impudicum. Molecular phylogeny was inferred using maximum likelihood and Bayesian inference with independent SSU and LSU rDNA sequences. Our results support the classification of Wangodinium within the Gymnodiniales sensu stricto clade and it is close to Lepidodinium. Our results also support the close relationship among G. dorsalisulcum, G. impudicum, and Barrufeta. Further research is needed to assign these Gymnodinium species to Barrufeta or to erect new genera.

First record of resting cysts of the benthic dinoflagellate Prorocentrum leve in a natural reservoir in Gujan-Mestras, Gironde, France.

The resting cysts of the benthic dinoflagellate Prorocentrum leve from a natural reservoir in Gujan-Mestras (Gironde, France) were described in this study. The incubated urn-shaped cysts gave rise to cells of P. leve. Morphological observations through light microscopy and scanning electron microscopy, particularly of the periflagellar platelets, combined with large subunit ribosomal DNA sequences obtained through single-cell analysis confirm their affinity to the species P. leve. The cysts were characterized by a specific shape and the presence of an anterior plug. This is the first conclusive evidence for fossilizable resting stages within the Prorocentrales, one of the major orders within the Dinophyceae. Palynological treatments show that the cysts and endospores withstand hydrochloric and hydrofluoric acids. Micro-Fourier transform infrared analysis on single specimens suggests that the composition of the endospore is cellulosic and the cyst wall a more robust, noncellulosic ß-glucan. The spectra overall are similar to other published spectra of resting cysts from autotrophic, planktonic dinoflagellates.

First report of fossilized cysts produced by the benthic Bysmatrum subsalsum (Dinophyceae) from a shallow Mexican lagoon in the Gulf of Mexico.

Cysts belonging to the benthic dinoflagellate Bysmatrum subsalsum were recovered from palynologically treated sediments collected in the Alvarado Lagoon (southwestern Gulf of Mexico). The cysts are proximate, reflecting the features of the parent thecal stage, and their autofluorescence implies a dinosporin composition similar to the cyst walls of phototrophic species. This finding is important for our understanding of B. subsalsum life cycle transitions and ecology. Encystment may play an important role in the bloom dynamics of this species as it can enable the formation of a sediment cyst bank that allows reinoculation of the water column when conditions become favorable. This is the first report of a fossilized cyst produced by a benthic dinoflagellate recovered from sub-recent sediments.

Relationship between the dinoflagellate cyst Spiniferites pachydermus and Gonyaulax ellegaardiae sp. nov. from Izmir Bay, Turkey, and molecular characterization.

Here, we established the cyst-motile stage relation-ship for Spiniferites pachydermus through incubation of cysts with a characteristically microreticulate/perforate surface isolated from Izmir Bay in the eastern Aegean Sea of the eastern Mediterranean. The morphology of the motile stage was similar to Gonyaulax spinifera but had a different size, overhang, displacement and reticulations. Based on the distinct morphology of the cyst and morphological differences in motile cells, we assigned S. pachydermus from Izmir Bay to the new species Gonyaulax ellegaardiae. We elucidate the phylogenetic relationship of G. ellegaardiae through large and small subunit ribosomal DNA and show that it forms a clade with other species that belong to the G. spinifera complex.

Cyst-motile stage relationship, morphology, ultrastructure, and molecular phylogeny of the gymnodinioid dinoflagellate Barrufeta resplendens comb. nov., formerly known as Gyrodinium resplendens, isolated from the Gulf of Mexico.

In the present study, we redescribed Gyrodinium resplendens through incubation of process bearing cysts extracted from sediment collected in the northern Gulf of Mexico. The morphology and ultrastructure of the motile stage and cyst stage were examined using light microscopy, scanning electron microscopy, and transmission electron microscopy and this revealed that the species should be transferred to the genus Barrufeta. This genus differs from other gymnodinioid genera in possessing a Smurf-cap apical structure complex (ASC) and currently encompasses only one species, Barrufeta bravensis. B. resplendens shows a Smurf-cap ASC that consists of three rows of elongated vesicles with small knobs in the middle one. B. resplendens is very similar to B. bravensis in cell morphology, but can be separated using the ultrastructure such as the shape and location of nucleus and pyrenoids, which highlights the importance of ultrastructure at inter-specific level in the genus Barrufeta. The unique cysts of B. resplendens are brown and process bearing, and have a tremic archeopyle with a zigzag margin on the dorsal side of the epicyst, and not polar as in cysts of Polykrikos. The cysts do not survive the palynological treatment used here and probably have a wide distribution. Maximum-likelihood and Bayesian inference were carried out based on partial large subunit ribosomal DNA (LSU rDNA) sequences. Molecular phylogeny supports that the genus Barrufeta is monophyletic, and that the genus Gymnodinium is polyphyletic. Our results suggest that details of the ASC together with ultrastructure are potential features to subdivide the genus Gymnodinium.

Differences in the chemical composition of organic-walled dinoflagellate resting cysts from phototrophic and heterotrophic dinoflagellates.

Dinoflagellates constitute a large proportion of the planktonic biomass from marine to freshwater environments. Some species produce a preservable organic-walled resting cyst (dinocyst) during the sexual phase of their life cycle that is an important link between the organisms, the environment in which their parent motile theca grew, and the sedimentary record. Despite their abundance and widespread usage as proxy indicators for environmental conditions, there is a lack of knowledge regarding the dinocyst wall chemical composition. It is likely that numerous factors, including phylogeny and life strategy, determine the cyst wall chemistry. However, the extent to which this composition varies based on inherent (phylogenetic) or variable (ecological) factors has not been studied. To address this, we used micro-Fourier transform infrared spectroscopy to analyze nine cyst species produced by either phototrophic or heterotrophic dinoflagellates from the extant orders Gonyaulacales, Gymnodiniales, and Peridiniales. Based on the presence of characteristic functional groups, two significantly different cyst wall compositions are observed that correspond to the dinoflagellate's nutritional strategy. The dinocyst wall compositions analyzed appeared carbohydrate-based, but the cyst wall produced by phototrophic dinoflagellates suggested a cellulose-like glucan, while heterotrophic forms produced a nitrogen-rich glycan. This constitutes the first empirical evidence nutritional strategy is related to different dinocyst wall chemistries. Our results indicated phylogeny was less important for predicting composition than the nutritional strategy of the dinoflagellate, suggesting potential for cyst wall chemistry to infer past nutritional strategies of extinct taxa preserved in the sedimentary record.

Toxic dinoflagellate Centrodinium punctatum (Cleve) F.J.R. Taylor: An examination on the responses in growth and toxin contents to drastic changes of temperature and salinity.

To understand environmental effects affecting paralytic shellfish toxin production of Centrodinium punctatum, this study examined the growth responses, and toxin contents and profiles of a C. punctatum culture exposed to drastic changes of temperature (5-30 °C) and salinity (15-40). C. punctatum grew over a temperature range of 15-25 °C, with an optimum of 20 °C., and over a salinity range of 25-40, with optimum salinities of 30-35. This suggests that C. punctatum prefers relatively warm waters and an oceanic habitat for its growth and can adapt to significant changes of salinity levels. When C. punctatum was cultivated at different temperature and salinity levels, the PST profile included four major analogs (STX, neoSTX, GTX1 and GTX4, constituted >80 % of the profile), while low amounts of doSTX and traces of dc-STX and dc-GTX2 were also observed. Interestingly, though overall toxin contents did not change significantly with temperature, increases in the proportion of STX, and decreases in proportions in GTX1 and GTX4 were observed with higher temperatures. Salinity did not affect either toxin contents or profile from 25 to 35. However, the total toxin content dropped to approximately half at salinity 40, suggesting this salinity may induce metabolic changes in C. punctatum.

Sudden peak in tetrodotoxin in French oysters during the summer of 2021: Source investigation using microscopy, metabarcoding and droplet digital PCR.

Tetrodotoxin (TTX) is a potent neurotoxin causing human intoxications from contaminated seafood worldwide and is of emerging concern in Europe. Shellfish have been shown to contain varying TTX concentrations globally, with concentrations typically higher in Pacific oysters Crassostrea gigas in Europe. Despite many decades of research, the source of TTX remains unknown, with bacterial or algal origins having been suggested. The aim of this study was to identify potential source organisms causing TTX contamination in Pacific oysters in French coastal waters, using three different techniques. Oysters were deployed in cages from April to September 2021 in an estuary where TTX was previously detected. Microscopic analyses of water samples were used to investigate potential microalgal blooms present prior or during the peak in TTX. Differences in the bacterial communities from oyster digestive glands (DG) and remaining flesh were explored using metabarcoding, and lastly, droplet digital PCR assays were developed to investigate the presence of Cephalothrix sp., one European TTX-bearing species in the DG of toxic C. gigas. Oysters analysed by liquid chromatography-tandem mass spectrometry contained quantifiable levels of TTX over a three-week period (24 June-15 July 2021), with concentrations decreasing in the DG from 424 µg/kg for the first detection to 101 µg/kg (equivalent to 74 to 17 µg/kg of total flesh), and trace levels being detected until August 13, 2021. These concentrations are the first report of the European TTX guidance levels being exceeded in French shellfish. Microscopy revealed that some microalgae bloomed during the TTX peak, (e.g., Chaetoceros spp., reaching 40,000 cells/L). Prokaryotic metabarcoding showed increases in abundance of Rubritaleaceae (genus Persicirhabdus) and Neolyngbya, before and during the TTX peak. Both phyla have previously been described as possible TTX-producers and should be investigated further. Droplet digital PCR analyses were negative for the targeted TTX-bearing genus Cephalothrix.

An unprecedented bloom of Lingulodinium polyedra on the French Atlantic coast during summer 2021.

At the end of July 2021, a bloom of Lingulodinium polyedra developed along the French Atlantic coast and lasted six weeks. The REPHY monitoring network and the citizen participation project PHENOMER contributed to its observation. A maximum concentration of 3,600,000 cells/L was reached on the 6th of September, a level never recorded on French coastlines. Satellite observation confirmed that the bloom reached its highest abundance and spatial extension early September, covering about 3200 km2 on the 4th of September. Cultures were established, and morphology and ITS-LSU sequencing identified the species as L. polyedra. The thecae displayed the characteristic tabulation and sometimes a ventral pore. The pigment composition of the bloom was similar to that of cultured L. polyedra, confirming that phytoplankton biomass was dominated by this species. The bloom was preceded by Leptocylindrus sp., developed over Lepidodinium chlorophorum, and was succeeded by elevated Noctiluca scintillans concentrations. Afterwards, relatively high abundance of Alexandrium tamarense were observed in the embayment where the bloom started. Unusually high precipitation during mid-July increased river discharges from the Loire and Vilaine rivers, which likely fueled phytoplankton growth by providing nutrients. Water masses with high numbers of dinoflagellates were characterized by high sea surface temperature and thermohaline stratification. The wind was low during the bloom development, before drifting it offshore. Cysts were observed in the plankton towards the end of the bloom, with concentrations up to 30,000 cysts/L and relative abundances up to 99%. The bloom deposited a seed bank, with cyst concentrations up to 100,000 cysts/g dried sediment, particularly in fine-grained sediments. The bloom caused hypoxia events, and concentrations of yessotoxins up to 747 µg/kg were recorded in mussels, below the safety threshold of 3,750 µg/kg. Oysters, clams and cockles also were contaminated with yessotoxins, but at lower concentrations. The established cultures did not produce yessotoxins at detectable levels, although yessotoxins were detected in the sediment. The unusual environmental summertime conditions that triggered the bloom, as well as the establishment of considerable seed banks, provide important findings to understand future harmful algal blooms along the French coastline.

Fensomea setacea, gen. & sp. nov. (Cladopyxidaceae, Dinophyceae), is neither gonyaulacoid nor peridinioid as inferred from morphological and molecular data.

Dinophyte evolution is essentially inferred from the pattern of thecal plates, and two different labelling systems are used for the important subgroups Gonyaulacales and Peridiniales. The partiform hypotheca of cladopyxidoid dinophytes fits into the morphological concepts of neither group, although they are assigned to the Gonyaulacales. Here, we describe the thecate dinophyte Fensomea setacea, gen. & sp. nov., which has a cladopyxidoid tabulation. The cells displayed a Kofoidean plate formula APC, 3', 4a, 7″, 7C, 6S, 6''', 2'''', and slender processes were randomly distributed over the echinate or baculate surface. In addition, we obtained rRNA sequences of F. setacea, gen. & sp. nov., but dinophytes that exhibit a partiform hypotheca did not show a close relationship to Gonyaulacales. Character evolution of thecate dinophytes may have progressed from the ancestral state of six postcingular plates, and two more or less symmetrically arranged antapical plates, towards patterns of only five postcingular plates (Peridiniales) or more asymmetrical configurations (Gonyaulacales). Based on our phylogenetic reconsiderations the contact between the posterior sulcal plate and the first postcingular plate, as well as the contact between an antapical plate and the distalmost postcingular plate, do not represent a rare, specialized gonyaulacoid plate configuration (i.e., the partiform hypotheca of cladopyxidoid dinophytes). Instead, these contacts correspond to the common and regular configuration of peridinioid (and other) dinophytes.

Sediment archives reveal irreversible shifts in plankton communities after World War II and agricultural pollution.

To evaluate the stability and resilience1 of coastal ecosystem communities to perturbations that occurred during the Anthropocene,2 pre-industrial biodiversity baselines inferred from paleoarchives are needed.3,4 The study of ancient DNA (aDNA) from sediments (sedaDNA)5 has provided valuable information about past dynamics of microbial species6-8 and communities9-18 in relation to ecosystem variations. Shifts in planktonic protist communities might significantly affect marine ecosystems through cascading effects,19-21 and therefore the analysis of this compartment is essential for the assessment of ecosystem variations. Here, sediment cores collected from different sites of the Bay of Brest (northeast Atlantic, France) allowed ca. 1,400 years of retrospective analyses of the effects of human pollution on marine protists. Comparison of sedaDNA extractions and metabarcoding analyses with different barcode regions (V4 and V7 18S rDNA) revealed that protist assemblages in ancient sediments are mainly composed of species known to produce resting stages. Heavy-metal pollution traces in sediments were ascribed to the World War II period and coincided with community shifts within dinoflagellates and stramenopiles. After the war and especially from the 1980s to 1990s, protist genera shifts followed chronic contaminations of agricultural origin. Community composition reconstruction over time showed that there was no recovery to a Middle Ages baseline composition. This demonstrates the irreversibility of the observed shifts after the cumulative effect of war and agricultural pollutions. Developing a paleoecological approach, this study highlights how human contaminations irreversibly affect marine microbial compartments, which contributes to the debate on coastal ecosystem preservation and restoration.

A new potentially toxic dinoflagellate Fukuyoa koreansis sp. nov. (Gonyaulacales, Dinophyceae) from Korean coastal waters: Morphology, phylogeny, and effects of temperature and salinity on growth.

To clarify an unspecified toxic Gambierdiscus-like species isolated from seawaters off Jeju Island, Korea, its morphology and molecular phylogeny based on the small subunit (SSU) and partial large subunit (LSU) rRNA gene sequences were examined. Cells were narrow in ventral view and broad in lateral view with a smooth surface. The round thecal pores were evenly distributed, with an average diameter of 0.41 µm. Cell depth, width and height were 51.7 ± 4.5 µm, 43.0 ± 4.2 µm and 55.0 ± 4.7 µm, respectively, and depth-to-width (D/W) and height-to-width (H/W) ratios were 1.1 ± 0.2 µm and 1.3 ± 0.02 µm, respectively. The nucleus was located in the hypotheca. Scanning electron microscope observations revealed that the cells displayed a plate formula of Po, 4', 6'', 6c, 6s, 5''' and 2''', and transmission electron microscope observation demonstrated that the cells contained crystal-like particles. Morphological features indicated that the unspecified Korean isolate belonged to the genus Fukuyoa, and based on the H/W and D/W ratios, the apical pore H/W ratio and thecal pore size, it could be differentiated from other Fukuyoa species. The phylogenetic analyses based on the SSU and LSU rRNA sequences revealed that the Korean isolate was nested within the genus Fukuyoa with high support, and it grouped with F. cf. yasumotoi isolated from Japan. Based on the morpho-molecular data, a new species, Fukuyoa koreansis sp. nov. is proposed. The maximum growth rate (0.254 d-1) of F. koreansis was observed at 25°C and a salinity of 25. The required levels of temperature and salinity for growth distinguished Fukuyoa koreansis from Gambierdiscus species.

Taxonomy and Molecular Phylogenetics of Ensiculiferaceae, fam. nov. (Peridiniales, Dinophyceae), with Consideration of their Life-history.

In the current circumscription, the Thoracosphaeraceae comprise all dinophytes exhibiting calcified coccoid cells produced during their life-history. Species hitherto assigned to Ensiculifera and Pentapharsodinium are mostly based on the monadoid stage of life-history, while the link to the coccoid stage (occasionally treated taxonomically distinct) is not always resolved. We investigated the different life-history stages and DNA sequence data of Ensiculifera mexicana and other species occurring in samples collected from all over the world. Based on concatenated ribosomal RNA gene sequences Ensiculiferaceae represented a distinct peridinalean branch, which showed a distant relationship to other calcareous dinophytes. Both molecular and morphological data (particularly of the coccoid stage) revealed the presence of three distinct clades within Ensiculiferaceae, which may include other dinophytes exhibiting a parasitic life-history stage. At a higher taxonomic level, Ensiculiferaceae showed relationships to parasites and endosymbionts (i.e., Blastodinium and Zooxanthella) as well as to dinophytes harbouring diatoms instead of chloroplasts. These unexpected phylogenetic relationships are corroborated by the presence of five cingular plates in all such taxa, which differs from the six cingular plates of most other Thoracosphaeraceae. We herein describe Ensiculiferaceae, emend the descriptions of Ensiculifera and Pentapharsodinium, erect Matsuokaea and provide several new combinations at the species level.

Centrodinium punctatum (Dinophyceae) produces significant levels of saxitoxin and related analogs.

Centrodinium punctatum is a fusiform dinoflagellate with a global marine distribution. Due to a close phylogenetic relationship of one C. punctatum strain to Alexandrium species, toxin production of this C. punctatum strain was assessed using liquid chromatography coupled to tandem mass spectrometry. The paralytic shellfish toxin (PST) profile of C. punctatum was dominated by six analogs, i.e. STX (30%), GTX-1 (20%) and neoSTX (24%), followed by GTX-2 (9%), GTX-4 (9%) and GTX-3 (8%); deoxy-STX was also putatively identified while no gymnodimines, spirolides or goniodomins were detected. This is the first record of C. punctatum producing saxitoxins. The estimated cellular toxicity was rather elevated, between 91 and 212 pg cell-1 (or 259 and 601 fmol cell-1). When considering the toxicity equivalent factors, results suggest that this species can produce high cellular toxicity compared to other STX-producing dinoflagellates. Morphological details of the sulcal area and the hypotheca of Centrodinium punctatum were re-examined by scanning electron microscopy (SEM); this revealed that in the sulcal area, the left posterior sulcal plate (Ssp) is larger and longer than the left posterior sulcal plate and extended into the hypotheca. Based on the morphological observation, a revised interpretation of the sulcus and hypotheca is proposed.

Morphological and phylogenetic data do not support the split of Alexandrium into four genera.

A recently published study analyzed the phylogenetic relationship between the genera Centrodinium and Alexandrium, confirming an earlier publication showing the genus Alexandrium as paraphyletic. This most recent manuscript retained the genus Alexandrium, introduced a new genus Episemicolon, resurrected two genera, Gessnerium and Protogonyaulax, and stated that: "The polyphyly [sic] of Alexandrium is solved with the split into four genera". However, these reintroduced taxa were not based on monophyletic groups. Therefore this work, if accepted, would result in replacing a single paraphyletic taxon with several non-monophyletic ones. The morphological data presented for genus characterization also do not convincingly support taxa delimitations. The combination of weak molecular phylogenetics and the lack of diagnostic traits (i.e., autapomorphies) render the applicability of the concept of limited use. The proposal to split the genus Alexandrium on the basis of our current knowledge is rejected herein. The aim here is not to present an alternative analysis and revision, but to maintain Alexandrium. A better constructed and more phylogenetically accurate revision can and should wait until more complete evidence becomes available and there is a strong reason to revise the genus Alexandrium. The reasons are explained in detail by a review of the available molecular and morphological data for species of the genera Alexandrium and Centrodinium. In addition, cyst morphology and chemotaxonomy are discussed, and the need for integrative taxonomy is highlighted.

Discovery of a New Clade Nested Within the Genus Alexandrium (Dinophyceae): Morpho-molecular Characterization of Centrodinium punctatum (Cleve) F.J.R. Taylor.

Investigation of phytoplankton from East China Sea of the Pacific Ocean, offshore Réunion Island of the Indian Ocean, and the French Atlantic coast revealed a species of poorly known armored fusiform dinoflagellate. To clarify this species, morphology and phylogeny based on mitochondrial and nuclear protein gene sequence (Cox1, Cob and Hsp90) concatenated with the SSU, ITS region and LSU rDNA sequences were analysed. Epifluorescence and scanning electron microscopy observations revealed that the nucleus of the specimen was elongated, sausage-shaped and located equatorially on the left lateral side of the cell, and that the plate formula is Po, 3', 1a, 6″, 6C, 8S, 5‴, 1p, 2'‴. These morphological features indicate that the species can be assigned to Centrodinium punctatum. Interestingly, the phylogenetic analyses placed this species within the Alexandrium clade, with Alexandrium affine being its closest relative. This indicates that genus Alexandrium is not monophyletic. The most similar morphological traits between C. punctatum and Alexandrium species were the shape of apical pore plate and the arrangement of the sulcal plates. However, since there are significant morphological differences between C. punctatum and Alexandrium species, further studies are needed to clarify the relation between the morphology and molecular phylogeny of other Centrodinium-related fusiform species.

Prorocentrum shikokuense Hada and P. donghaiense Lu are junior synonyms of P. obtusidens Schiller, but not of P. dentatum Stein (Prorocentrales, Dinophyceae).

In Japanese, Chinese and Korean coastal waters, recurrent blooms of a small, elongate Prorocentrum species have been observed in recent years. In previous studies, this species has been respectively identified as P. shikokuense, P. donghaiense and P. dentatum, despite morphological similarity and identical rDNA sequences. To resolve the confusion, morphological features, including the architectural details of the periflagellar area, were examined and ribosomal DNA (rDNA) sequences were obtained from specimens collected from the East China Sea and Korean coast, and a strain established in the waters off the Canary Islands of Spain. In addition, the descriptions of the three species and allied species were reviewed. Morphological observations and a phylogeny based on the SSU, ITS region and LSU rDNA sequences revealed that the previously confused species and our studied strains are conspecific and that the morphology of strains identified as P. dentatum in the phylogenetic trees does not coincide with P. dentatum sensu stricto. The confusion can be traced back to Dodge (1975, p. 116), who considered P. veloi, P. monacense and P. obtusidens as junior heterotypic synonyms of P. dentatum. However, Dodge's P. dentatum are closer to P. obtusidens sensu stricto, rather than P. dentatum sensu stricto. P. obtusidens sensu stricto can be distinguished from P. dentatum sensu stricto by its relatively small size, parallel sides towards the anterior and a blunt anterior extension on one side. This indicates that P. obtusidens should not be considered a synonym of P. dentatum sensu stricto. In addition, a comparison of the original descriptions of P. obtusidens and allied species allowed to conclude that small, elongate Prorocentrum from Japanese, Chinese and Korean coastal waters previously identified as P. shikokuense, P. donghaiense, P. dentatum sensu Yoo and Lee (1986) and the specimens studied herein, which share identical rDNA sequences, morphologically coincide with P. obtusidens. Therefore, it is proposed that P. shikokuense and P. donghaiense should be regarded as junior synonyms of P. obtusidens.