Azadinium Species Research Articles

A new small thecate dinoflagellate Azadinium anteroporum sp. nov. (Amphidomataceae, Dinophyceae) isolated from the Asian Pacific

ABSTRACT The marine thecate dinoflagellate Azadinium includes species known to produce the diarrhetic shellfish toxins known as azaspiracids (AZAs). In this study, the morphology of a thecate dinoflagellate isolated from Mutsu Bay, Japan, was examined by LM and SEM, and its phylogenetic position was inferred from ITS and LSU rDNA sequences. The production of AZAs was examined by liquid chromatography-triple quadrupole mass spectrometer (LC-MS/MS). Cells were elliptical, 15.9–22.4 µm long and 11.1–17.1 µm wide. A chloroplast was positioned peripherally, with a pyrenoid near the cingulum, and the nucleus was placed in the hyposome. The hyposome was smaller than the episome, with an antapical spine on plate 2''''. Thecal tabulation was Po, cp, X, 4', 3a, 6'', 6C, 5S, 6''', 2''''. The apical pore complex consisted of a symmetrical apical pore plate (Po), a cover plate (cp) and a small canal plate (X), and was surrounded by a prominent collar formed by the apical plates. A ventral pore was located on the mid-anterior edge of plate 1' and contacted with the X-plate and/or the ventral left of plate Po. Plate 1'' was in contact with plate 1a. AZA was not detected in the cultures. Molecular phylogeny showed that the species was related to Azadinium species that possess the ventral pore on the left side of plate 1', i.e. A. cuneatum, A. dalianense, A. obesum, A. poporum, A. spinosum and A. trinitatum, but that it was not included in these species. The new species Azadinium anteroporum sp. nov. differs in the ventral pore position (mid-anterior edge of plate 1') from these related species (which have the ventral pore on the left side of plate 1'), and from A. polongum (with ventral pore on the posterior left side of 1') and all other Azadinium species (with ventral pore on the right side of 1').

Metabarcoding revealed a high diversity of Amphidomataceae (Dinophyceae) and the seasonal distribution of their toxigenic species in the Taiwan Strait

The dinophyte family Amphidomataceae includes the genera Azadinium and Amphidoma. Four of these species are known to produce azaspiracids, which are lipophilic phycotoxins accumulating in shellfish. The diversity and biogeography of Amphidomataceae is far from yet resolved. Here we performed a time series sampling of both water and sediments in the Taiwan Strait from Nov. 2018 to April 2021. Metabarcoding was performed to unveil the diversity of Amphidomataceae targeting internal transcribed spacer (ITS1) region and partial large subunit ribosomal DNA (LSU rDNA D1–D3), followed by quantitative PCR (qPCR) with modified primers for Az. poporum ribotypes. The diversity of Amphidomataceae was revealed from the water samples with the aid of ITS1 and LSU based molecular phylogeny. The LSU based approach detected only a few species. In contrast, ITS1 based dataset showed eight new Azadinium clades and several ZOTUs (zero-radius operational taxonomic units) grouping together with Am. languida. Moreover, eleven known Azadinium species including three ribotypes of Az. poporum and Az. dexteroporum, and two ribotypes of Az. spinosum, were detected. The latter two species have not been reported in China before. Among these toxigenic species, Az. poporum was relevantly abundant whereas others were rare. The maximum of 209 cells L −1 of Az. poporum ribotype A was estimated using qPCR nearby Quanzhou in Nov. 2018 and 172 cells L 1 of Az. poporum ribotype B was detected far off coast in Apr. 2021. Metabarcoding on sediment samples revealed Az. poporum ribotypes B and C, but strains obtained with sediment incubation experiments yielded only ribotype B. Using qPCR about 0.2 cysts g −1 of Az. poporum ribotype B were quantified in May 2019 but cysts of Az. poporum ribotype C were not detected. Our results suggest that metabarcoding targeting ITS1 region is powerful to uncover the diversity of harmful dinophytes. Our results also highlight the rich diversity of Amphidomataceae and risk potential of azaspiracids in the Taiwan Strait and surrounding waters.

Increase in records of toxic phytoplankton and associated toxins in water samples in the Patagonian Shelf (Argentina) over 40 years of field surveys

Historical records (1980–2018) of potentially toxic phytoplankton and phycotoxins on the Argentine Continental Shelf (35°S-56.5°S) and adjacent ocean waters were systematically reviewed from scientific literature to assess their abundance and diversity over spatial and temporal scales. Records increased from 124 in the period 1980–1992 to 638 in 2006–2018, and the scanned area expanded from coastal to offshore waters including the shelf-break front. Alexandrium was the most reported genus (54%) during 1980–1992 and Pseudo-nitzschia (52%) during 1993–2005. By 2006–2018, a higher diversity was documented: Alexandrium (20%), Dinophysis (32%), Pseudo-nitzschia (31%), and the most recently described potentially toxic dinoflagellates of the family Amphidomataceae (8%). Likewise, a wider spectrum of phycotoxins was documented in the last decade, with lipophilic (LSTs) and paralytic shellfish toxins (PSTs) as the most recorded. Increased records are related to intensified monitoring, more detailed taxonomic analyses and more sensitive chemical techniques for marine biotoxin detection. This quantitative assessment brings light to the widespread occurrence of HABs along contrasting areas of the Patagonian Shelf and sets the basis for ecosystem risk evaluation. Moreover, comparison of toxic phytoplankton reported in the SW Atlantic with those in similar temperate seas in the North Atlantic and the Pacific Ocean, disclose ocean basin differences in strain toxicity of A. ostenfeldii, D. tripos and Azadinium species.

Insights into the discrepancy between Azadinium spp. and azaspiracid toxins near strategically important aquaculture operations in the west and southwest of Ireland

Apparent increases in harmful algal blooms worldwide have fostered attempts at mitigating their impacts on the aquaculture industry. The dinoflagellate species Azadinium spinosum has been described as the de novo azaspiracid (AZA) toxin producer of AZA-1 and -2 and been implicated in shellfish poisoning incidents (AZP) around Europe, regularly affecting shellfish mariculture operations.Several species of Azadinium have been confirmed in Irish coastal waters, and routine monitoring has shown disparities between Azadinum spp. cell count estimates in the water column and AZA concentrations in shellfish.A survey of bays on the southwest and west coasts of Ireland, carried out in August 2016, investigated the late summer distribution of Azadinium spp. and AZAs. Molecular analysis of water column samples showed very low levels of Az. spinosum. However, AZAs were found in 44% of samples, including in Az. spinosum negative samples. PCR-DGGE analysis was carried out using Amphidomataceae family specific primers on a selection of Az. spinosum negative, but AZA positive, samples. Subsequently sequenced DNA amplicons showed a high level of similarity with other Azadinium species suggesting that the species-specific molecular assays, in current use for monitoring Azadinium spp., are not capturing the likely greater diversity of the genus in Irish waters.

Toxigenic strains of Azadinium poporum (Amphidomataceae, Dinophyceae) from Japan and Vietnam, with first reports of A. poporum (ribotype A) and A. trinitatum in Asian Pacific

SUMMARYMorphology and phylogeny of small armored dinoflagellate Azadinium species isolated from Japanese and Vietnamese coasts were examined by light microscopy, scanning electron microscopy and molecular phylogeny inferred from ITS and LSU rDNA (D1–D3) sequences. Dominant azaspiracids (AZAs) were analyzed by using liquid chromatography/triple quadrupole mass spectrometer (LC/MS/MS). Sixteen and four culture strains were established from Japan and Vietnam, respectively. A phylogenetic tree confirmed our species identifications of Japanese cultures as Azadinium poporum (ribotypes A, B and C), A. trinitatum and A. zhuanum, and Vietnamese cultures as A. poporum (ribotype B). Typical cells of A. poporum had the thecal tabulation of Po, cp, X, 4′, 3a, 6″, 6C, 5S, 6″′, 2″″, A. trinitatum had Po, cp, X, 4–5′, 3a, 6–7″, 6C, 5S, 5–6″′, 2″″ and A. zhuanum had Po, cp, X, 3–4′, 2a, 6″, 6C, 5S, 6″′, 2″″. Cellular morphology of the three species were congruent with those in the original reports, in terms of the presence/absence of antapical spine and the position of the ventral pore, nucleus and pyrenoids, although small differences were also found in the nucleus position of A. trinitatum and A. zhuanum. AZA‐2 was the dominant component in Japanese A. poporum (ribotypes A, B and C), and AZA‐40 in Vietnamese A. poporum (ribotype B). AZA‐2 and AZA‐40 were not detected in Vietnamese and Japanese A. poporum, respectively. AZA analogs were not detected from A. trinitatum and A. zhuanum. This is the first report of the toxigenic A. poporum from Japan and Vietnam, with identifications of the major AZAs. The ribotype A of A. poporum and A. trinitatum were the first records in Asia.

Morphological and molecular characterization of multiple new Azadinium strains revealed a high diversity of non‐toxigenic species of Amphidomataceae (Dinophyceae) including two new Azadinium species in Irish waters, North East Atlantic

SUMMARYShellfish contamination with azaspiracids (AZA), which are lipophilic marine biotoxins produced by marine dinoflagellates, is a major and recurrent problem for the Irish shellfish industry. AZA are produced by certain species of Amphidomataceae, but the species diversity of this group of microalgae in Irish waters is poorly known. Here we present a morphological and molecular characterization of multiple new strains of non‐toxigenic Azadinium isolated on an oceanographic survey in 2018. A lack of AZA production for all strains presented here was demonstrated by LC‐MS/MS analysis. One strain of Azadinium caudatum var. margalefii (first strain for the area) confirmed non‐toxigenicity of Atlantic populations of this species. One strain designated as Azadinium cf. zhuanum was similar to Az. zhuanum described from China but differed from the type strain in nucleus position, by the dominant number of apical plates, and by significant differences in rRNA gene sequences. Finally, two new non‐toxigenic Azadinium species are described from the North East Atlantic: Azadinium galwayense sp. nov. and Azadinium perfusorium sp. nov. Azadinium galwayense differed from other Azadinium by a characteristic combination regarding presence and location of the ventral pore (vp; on the right side of the pore plate), of a pyrenoid (located in the episome), and by a pentagonal shape of the median anterior intercalary plate 2a, and lack of contact between plates 1″ and 1a. Azadinium perfusorium shared the same vp position as Az. galwayense and differed by a characteristic combination of a pyrenoid located in the hyposome, a tetragonal shape of plate 2a, and a relatively large size of the two lateral anterior intercalary plates. Molecular phylogeny confirmed the distinctiveness of these two new species and their placement in Azadinium. The present findings significantly increased knowledge on the diversity of Azadinium species in the North East Atlantic.

Temporal and spatial distribution of Azadinium species in the inland and coastal waters of the Pacific northwest in 2014–2018

Azaspiracids, produced by some species of the dinoflagellate genera Azadinium and Amphidoma, can cause a syndrome in humans called azaspiracid shellfish poisoning (AZP). In 1995, mussels from the Irish west coast contaminated with azaspiracids were, for the first time, linked to this human illness that has symptoms of nausea, vomiting, severe diarrhea, and stomach cramps. The only confirmed cases of AZP to date in the United States occurred in Washington State in 2008 from mussels imported from Ireland. Shortly after this case, several others involving similar gastrointestinal symptoms were reported by shellfish consumers from Washington State. However, no detectable diarrhetic shellfish toxins or Vibrio contamination were found. Cursory analysis of Solid Phase Adsorption Toxin Tracking (SPATT) samplers suggested the presence of azaspiracids in Washington State waters and motivated a study to evaluate the presence and distribution of Azadinium species in the region. During the spring and summer months of 2014–2015, quantitative polymerase chain reaction (qPCR) analyses detected the presence of the toxigenic species Azadinium poporum and A. spinosum on the outer coast and throughout the inland waters of Washington State. In 2016–2018, standard curves developed using A. poporum isolated from Puget Sound and A. spinosum isolated from the North Sea were used to quantify abundances of up to 10,525 cells L−1 of A. poporum and 156 cells L−1 of A. spinosum at shore-based sites. Abundances up to 1,206 cells L−1 of A. poporum and 30 cells L−1 of A. spinosum were measured in the coastal waters of the Pacific Northwest in 2017. Other harmful genera, including Alexandrium, Dinophysis, and Pseudo-nitzschia, were observed using light microscopy at coastal sites where A. poporum was also observed. In some samples where both A. poporum and A. spinosum were absent, an Amphidomataceae-specific qPCR assay indicated that other species of Azadinium or Amphidoma were present. The identification of Azadinium species in the PNW demonstrates the need to assess their toxicity and to incorporate their routine detection in monitoring programs to aid resource managers in mitigating risks to azaspiracid shellfish poisoning in this region.

Amphidomataceae (Dinophyceae) in the western Greenland area, including description of Azadinium perforatum sp. nov.

ABSTRACTAzaspiracids (AZA) are lipophilic marine biotoxins associated with shellfish poisoning which are produced by some species of Amphidomataceae. Diversity and global biogeography of this family are still poorly known. In summer 2017 plankton samples were collected from the central Labrador Sea and western Greenland coast from 64° N (Gothaab Fjord) to 75° N for the presence of Amphidomataceae and AZA. In the central Labrador Sea, light microscopy revealed small Azadinium-like cells (9200 cells l−1). Clonal strains established from plankton samples and scanning electron microscopy of fixed plankton samples revealed at least eight species of Amphidomataceae: Azadinium obesum, Az. trinitatum, Az. dexteroporum, Az. spinosum, Az. polongum, Amphidoma languida, Azadinium spec., and a new species described here as Azadinium perforatum sp. nov. The new species differed from other Azadinium species by the presence of thecal pores on the pore plate. All samples, including cultured strains, filtered seawater samples, and solid phase adsorption toxin tracking (SPATT) samplers deployed during the expedition in a continuous water-sampling system (FerryBox), were negative for AZA. DNA samples and PCR assays were positive for Amphidomataceae from most stations, whereas species-specific assays for three toxigenic species were rarely positive (two stations for Az. poporum, one station for Am. languida). The results highlight the presence of Amphidomataceae in the area but the lack of toxins and low abundance of toxigenic species currently indicate a low risk of toxic Amphidomataceae blooms in Arctic coastal waters.

Field observations of the dinoflagellate genus Azadinium and azaspiracid toxins in the south-west Atlantic Ocean

Some dinoflagellate species of the genera Azadinium and Amphidoma (Amphidomataceae) produce azaspiracids (AZA), a group of toxins responsible for gastrointestinal disorders in humans following the consumption of contaminated shellfish. In this study, we investigated the diversity, distribution and abundance of Azadinium and AZA from field plankton samples collected during four oceanographic expeditions that covered an extended area of the Argentine Sea during different seasons. Scanning electron microscopy analyses indicated the presence of five Azadinium species: Az. dexteroporum, Az. luciferelloides, Az. obesum, Az. asperum and Az. cf. poporum. Azadinium-like cells were frequently found and were even an abundant component of plankton assemblages, showing a wide latitudinal distribution, from ~38 to ~55.5°S, and occurring in a wide temperature and salinity range. High cell densities (up to 154000cellsL–1) occurred in northern slope and external shelf waters during spring. AZA-2 was detected in net samples from the 20- to 200-µm fractions by tandem mass spectrometry–liquid chromatography analysis, suggesting a transfer of AZA through the food web. Our results contribute to the knowledge of the worldwide occurrence of Azadinium species and AZA, and highlight the importance of amphidomatacean species as a potential source of AZA shellfish poisoning in the south-west Atlantic Ocean.

First detection of species of the potentially toxic genus Azadinium (Amphidomataceae, Dinophyceae) in tropical coastal waters of Brazil

In fall 1995, during a survey in Abrolhos coral reef system (southwestern Atlantic, Brazil), significant densities (143–6174 cells L−1) of small thecate dinoflagellates were detected. Analysis of this material in scanning and transmission electron microscopy confirmed the presence of four taxa assigned to the potentially toxic genus Azadinium: A. dexteroporum Percopo et Zingone, A. luciferelloides Tillmann et Akselman, A. cf. polongum Tillmann and Azadinium sp. The latter taxon showed external morphological features quite distinct from any Azadinium taxon yet described, but its formal description as a new species depends on more detailed analysis. Species of Azadinium have never been confirmed in Brazilian waters until now, although the toxins produced by these dinoflagellates, the azaspiracids, have been detected in Brazilian southern coast without recognition of their producing organisms. The highest densities of Azadinium spp. occurred at stations south of and over the Abrolhos Bank, which receive higher nutrient concentrations due to upwelling of deep and nutrient-rich water masses.

Identification of Azadinium species and a new azaspiracid from Azadinium poporum in Puget Sound, Washington State, USA

The identification of a new suite of toxins, called azaspiracids (AZA), as the cause of human illnesses after the consumption of shellfish from the Irish west coast in 1995, resulted in interest in understanding the global distribution of these toxins and of species of the small dinoflagellate genus Azadinium, known to produce them. Clonal isolates of four species of Azadinium, A. poporum, A. cuneatum, A. obesum and A. dalianense were obtained from incubated sediment samples collected from Puget Sound, Washington State in 2016. These Azadinium species were identified using morphological characteristics confirmed by molecular phylogeny. Whereas AZA could not be detected in any strains of A. obesum, A. cuneatum and A. dalianense, all four strains of A. poporum produced a new azaspiracid toxin, based on LC–MS analysis, named AZA-59. The presence of AZA-59 was confirmed at low levels in situ using a solid phase resin deployed at several stations along the coastlines of Puget Sound. Using a combination of molecular methods for species detection and solid phase resin deployment to target shellfish monitoring of toxin at high-risk sites, the risk of azaspiracid shellfish poisoning can be minimized.

Adding new pieces to the Azadinium (Dinophyceae) diversity and biogeography puzzle: Non-toxigenic Azadinium zhuanum sp. nov. from China, toxigenic A. poporum from the Mediterranean, and a non-toxigenic A. dalianense from the French Atlantic

The marine planktonic dinophyceaen genus Azadinium is a primary source of azaspiracids, but due to their small size its diversity may be underestimated and information on its biogeography is still limited. A new Azadinium species, A. zhuanum was obtained from the East China Sea and Yellow Sea of China by incubating surface sediments. Five strains were established by isolating single germinated cells and their morphology was examined with light microscopy and scanning electron microscopy. Azadinium zhuanum was characterized by a plate pattern of Po, cp, X, 4′, 2a, 6′′, 6C, 5S, 6′′′, 2′′′′, by a distinct ventral pore at the junction of Po, the first and fourth apical plates, and a conspicuous antapical spine. Moreover, Azadinium poporum was obtained for the first time from the Mediterranean by incubating surface sediment collected from Diana Lagoon (Corsica) and a new strain of Azadinium dalianense was isolated from the French Atlantic. The morphology of both strains was examined. Small subunit ribosomal DNA (SSU rDNA), large subunit ribosomal DNA (LSU rDNA) and internal transcribed spacer (ITS) sequences were obtained from cultured strains. In addition, LSU sequences were obtained by single cell sequencing of two presumable A. poporum cells collected from the French Atlantic. Molecular phylogeny based on concatenated SSU, LSU and ITS sequences revealed that A. zhuanum was closest to A. polongum. French A. poporum from Corsica (Mediterranean) and from the Atlantic showed some genetic differences but were nested within one of the A. poporum ribotypes together with other European strains. Azadinium dalianense from France together with the type strain of the species from China comprised a well resolved clade now consisting of two ribotypes. Azaspiracid profiles were analyzed for the cultured Azadinium strains using LC–MS/MS and demonstrate that the Mediterranean A. poporum strain produced AZA-2 and AZA-2 phosphate with an amount of 0.44fgcell−1. Azadinium zhuanum and A. dalianense did not produce detectable AZA. Results of the present study support the view of a high diversity and wide distribution of species belonging to Azadinium. The first record of AZA-2 producing A. poporum from the Mediterranean suggests that this species may be responsible for azaspiracid contaminations in shellfish from the Mediterranean Sea.

Revisiting the 1991 algal bloom in shelf waters off Argentina: Azadinium luciferelloides sp. nov. (Amphidomataceae, Dinophyceae) as the causative species in a diverse community of other amphidomataceans

SUMMARYSpecies of the marine dinophycean genera Azadinium and Amphidoma (Amphidomataceae) mostly attract attention because of their production potential of the lipophilic polyether phycotoxin azaspiracid (AZA). The genus Azadinium probably has a very wide geographical distribution. Blooms of Azadinium from the continental shelf off Argentina have been observed back in the early 1990, but were just recently published, and the causative species, identified at that time as Azadinium cf. spinosum, could not unequivocally be determined. Here we retrospectively analyzed old archived samples of one of the South Atlantic Azadinium bloom from 1991 with electron microscopy. It turned out that the dominant nanoplanktonic dinophycean species in fact represent a new species which we describe here based on the morphology. Azadinium luciferelloides sp. nov. is a small (approximately 9–14 μm cell length) thecate dinoflagellate with the dominant plate pattern of the genus (Po, X, 4′, 3a, 6″, 6C, 5S, 6″′, 2″″), and with a small antapical spine. Azadinium luciferelloides differed from all other described species of Azadinium by the position of the ventral pore, which was located on the right ventral side in a notch of an otherwise symmetric pore plate. In addition, we recorded and documented the presence of other similar sized species of the Amphidomataceae in the samples. Our finding of Az. spinosum, Az. dalianense, Az. dexteroporum, and Amphidoma languida are the first record for the South Atlantic and thus describe an important range extension of these species. The diversity and importance of the Amphidomataceae for South Atlantic spring bloom plankton is now known and taxonomically documented, but cultures and/or analysis of AZA in field samples of the area are needed to clarify the AZA production potential of the local species and populations in order to finally evaluate the risk potential of AZA for AZA shellfish contamination in the Southwestern Atlantic region.

Azadinium dalianense, a new dinoflagellate species from the Yellow Sea, China

Luo Z., Gu H., Krock B. and Tillmann U. 2013. Azadinium dalianense, a new dinoflagellate species from the Yellow Sea, China. Phycologia 52: 625–636. DOI: 10.2216/13-178.1The dinoflagellate genus Azadinium includes species with a plate formula of po, cp, X, 4′, 3a, 6′′, 6C, 5S, 6′′′, 2′′′′ and is part of the family Amphidomataceae with an uncertain order affiliation. Among six species, at least two produce azaspiracids (AZAs), a group of lipophilic toxins that accumulate in shellfish and can cause human health problems. Diversity within the genus might be underestimated at present due to its small size. In the present study, we searched for Azadinium by incubating freshly collected sediments from the Yellow Sea off China and succeeded in detecting a new species, here described as Azadinium dalianense sp. nov. It shared identical hypothecal, cingular and sulcal plates with the other Azadinium species, but it was unique in having only three apical and two anterior intercalary plates. Up to two stalked pyrenoids were present but their location in the cell varied. Phylogenetic analyses based on concatenated small-subunit, partial large-subunit, and internal transcribed spacer sequences revealed that A. dalianense was nested within Azadinium and formed a strongly supported clade with A. poporum. Liquid chromatography–mass spectrometry analyses did not detect any known AZAs.

A new potentially toxic Azadinium species (Dinophyceae) from the Mediterranean Sea, A.dexteroporum sp. nov.

A new photosynthetic planktonic marine dinoflagellate, Azadinium dexteroporum sp. nov., is described from the Gulf of Naples (South Tyrrhenian Sea, Mediterranean Sea). The plate formula of the species, Po, cp, X, 4', 3a, 6″, 6C, 5?S, 6‴ and 2″″, is typical for this recently described genus. Azadinium dexteroporum is the smallest rep-resentative of the genus (8.5μm average length, 6.2μm average width) and shares the presence of a small antapical spine with the type species A.spinosum and with A.polongum. However, it differs from all other Azadinium species for the markedly asymmetrical Po plate and the position of the ventral pore, which is located at the right posterior end of the Po plate. Another peculiarity of A.dexteroporum is the pronounced concavity of the second intercalary plate (2a), which appears collapsed with respect to the other plates. Phylogenetic analyses based on the large subunit 28S rDNA (D1/D2) and the internal transcribed spacer (ITS rDNA) support the attribution of A.dexteroporum to the genus Azadinium and its separation from the other known species. LC/MS-TOF analysis shows that Azadinium dex-teroporum produces azaspiracids in low amounts. Some of them have the same molecular weight as known compounds such as azaspiracid-3 and -7 and Compound 3 from Amphidoma languida, as well as similar fragmentation patterns in some cases. This is the first finding of a species producing azapiracids in the Mediterranean Sea.

TAXONOMIC REVISION OF THE DINOFLAGELLATE AMPHIDOMA CAUDATA: TRANSFER TO THE GENUS AZADINIUM (DINOPHYCEAE) AND PROPOSAL OF TWO VARIETIES, BASED ON MORPHOLOGICAL AND MOLECULAR PHYLOGENETIC ANALYSES(1).

The systematic position of Amphidoma caudata Halldal within the genus Amphidoma has remained uncertain as a result of its plate formula and the absence of molecular phylogenetic data. Also, this thecate dinoflagellate taxon has been used to designate two distinct morphotypes. The present study aims to clarify the generic affiliation of Amphidoma caudata and the taxonomic value of two different morphotypes M1 and M2. The new examination of the plate formula using SEM showed that it was the same for both morphotypes and that it corresponded to the tabulation of the recent erected genus Azadinium Elbrächter et Tillmann. Morphometric analysis, using cell size, length of apical projection in conjunction with the cell length, and the ratio of horn and spine showed that M1 and M2 formed two distinct groups. These results were supported by a molecular approach, revealing notable differences in the sequences of LSU rDNA and ITS region between these two morphotypes. Phylogenetic analyses inferred either from LSU and combined SSU, ITS region and COI data positioned M1 and M2 in a sister cluster of Azadinium species while Amphidoma languida Tillmann, Salas et Elbrächter, the only species of Amphidoma for which sequence data were available, was situated in a basal position of the Azadinium clade. Thus, we propose the transfer of Amphidoma caudata to the genus Azadinium and, consequently, the rehabilitation of the original tabulation of the genus Amphidoma Stein. To discriminate the two morphotypes, we propose a rank of variety with the following designations: Azadinium caudatum var. caudatum and Azadinium caudatum var. margalefii.

First Report of the Photosynthetic Dinoflagellate Genus Azadinium in the Pacific Ocean: Morphology and Molecular Characterization of Azadinium cf. poporum

A strain of a dinoflagellate belonging to the genus Azadinium was obtained by the incubation of sediments collected from Shiwha Bay, Korea. This report of the genus Azadinium is the first outside of northern Europe and furthermore from the Pacific Ocean. The diagnostic morphological features of the isolate very closely resemble the recently described species Azadinium poporum isolated from the North Sea. However, the shape of the 3' apical plate and the occasional morphological variations unreported from A. poporum bring minor distinctions between strains from different locations. The DNA sequences of small subunit, ITS, and large subunit (LSU) rDNA differed by 0.2%, 2.6%, and 3.6%, respectively, from those of A. poporum, whereas the COI gene was identical to those found in all strains of Azadinium. Phylogenetic analyses of the ribosomal DNA regions generally positioned the Korean strain as a sister taxon of A. poporum. However, the Korean isolate tends to occupy a basal position within Azadinium species with ITS rDNA and LSU rDNA. Using liquid chromatography coupled with tandem mass spectrometry, no known azaspiracids were detected. The slight but discernible morphological differences, the distinct rDNA sequences, and the tendency of the Korean strain to diverge phylogenetically based on ITS rDNA and LSU rDNA from A. poporum do not enable us to clearly assign the isolate to A. poporum. However, these characteristics do not allow us to classify it as a distinct species, and it is therefore designated as Azadinium cf. poporum. The examination of more strains to find more diagnostic characteristics might enable the attribution of this material to a well-defined taxonomic position.

A new non-toxic species in the dinoflagellate genus Azadinium: A. poporum sp. nov.

A new dinoflagellate species, Azadinium poporum sp. nov., was isolated as three clones from the southern North Sea off the Danish coast. In contrast to the type species A. spinosum, a known producer of azaspiracid (AZA) toxins, the isolates of A. poporum produce no known AZA analogues detectable by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Azadinium poporum is a small (11–16 µm length; 8–12 µm width) photosynthetic dinoflagellate with a thin theca exhibiting the Kofoidean plate tabulation: Po, cp, X, 4′, 3a, 6″. 6C, 5?S, 6′″, 2″″. This species is morphologically distinguished from the type A. spinosum by a slightly lower mean cell length/width ratio, consistent absence of an antapical spine, the presence of more than one stalked pyrenoid, and the conspicuous arrangement of the ventral pore, located at the junction of the pore plate and the first two apical plates. This latter feature also distinguishes A. poporum from A. obesum. As in A. spinosum, but differing from A. obesum, the first precingular (1″) plate of A. poporum touches the first epithecal intercalary plate 1a. DNA sequences and phylogenetic analysis of four nuclear-encoded genes, namely the internal transcribed spacer (ITS), 18S rDNA, 28S rDNA (D1/D2) and cytochrome oxidase I (COI), support the separation of A. poporum from (but with a close affinity to) other Azadinium species (A. obesum and A. spinosum), as well as its description as a distinct species within the recently erected genus Azadinium Elbrächter & Tillmann. Nevertheless, in spite of the phylogenetic consistency at the generic level, the position of the genus with respect to higher taxonomic levels within the subclass Peridiniphycidae could not be further clarified.