Morphology and molecular phylogeny of the benthic dinoflagellates (Dinophyceae, Peridiniales) Amphidiniopsis crumena n. sp. and Amphidiniopsis nileribanjensis n. sp.

Amphidiniopsis is a benthic, heterotrophic and thecate dinoflagellate genus that has a smaller epitheca and larger hypotheca. The genus contains 24 described species, but is considered to be polyphyletic based on morphological characters and molecular phylogenetics. In this study, two new species were discovered from two distant sampling localities, Amphidiniopsis crumena sp. nov. from Japan, and Amphidiniopsis nileribanjensis sp. nov., from Australia. These species have a uniquely shaped, additional second postcingular plate. Both species are dorsoventrally flattened, an apical hook is present, and have six postcingular plates. The plate formula is: APC 4' 3a 7″ ?C 4?S 6″' 2″″. The cells of these species were examined with LM and SEM, and molecular phylogenic analyses were performed using 18S and 28S rDNA. These species are distinguished by the presence of spines on the hypotheca and touching of the sixth postcingular plate and the anterior sulcal plate. Their shape and disposition of several thecal plates also differ. Molecular phylogenetic analyses showed that the two new species formed a monophyletic clade and did not belong to any morphogroup proposed by previous studies. Considering the morphological features and the molecular phylogenetic results, a new morphogroup is proposed, Amphidiniopsis morphogroup VI ('crumena group').

Geographical distribution, molecular and toxin diversity of the dinoflagellate species Gambierdiscus honu in the Pacific region.

An increase in cases of ciguatera poisoning (CP) and expansion of the causative species in the South Pacific region highlight the need for baseline data on toxic microalgal species to help identify new areas of risk and manage known hot spots. Gambierdiscus honu is a toxin producing and potential CP causing dinoflagellate species, first described in 2017. Currently no high-resolution geographical distribution, intraspecific genetic variation or toxin production diversity data is available for G. honu. This research aimed to further characterize G. honu by investigating its distribution using species-specific real-time polymerase chain reaction assays at 25 sites in an area spanning ∼8000 km of the Coral Sea/Pacific Ocean, and assessing intraspecific genetic variation, toxicity and toxin production of isolated strains. Assessment of genetic variation of the partial rRNA operon of isolates demonstrated no significant intraspecific population structure, in addition to a lack of adherence to isolation by distance (IBD) model of evolution. The detected distribution of G. honu in the Pacific region was within the expected tropical to temperate latitudinal ranges of 10° to -30° and extended from Australia to French Polynesia. In the lipophilic fractions, the neuroblastoma cell-based assay (CBA-N2a) showed no ciguatoxin (CTX)-like activity for nine of the 10 isolates, and an atypical pattern for CAWD233 isolate which showed cytotoxic activity in OV- and OV+ conditions. In the same way, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis confirmed no Pacific-CTXs (CTX-3B, CTX-3C, CTX-4A, CTX-4B) were produced by the ten strains. The CBA-N2a assessment of the hydrophilic fractions showed moderate to high cytotoxicity in both OV- and OV+ condition for all the strains showing a cytotoxic profile similar to that of gambierone. Indeed, this study is the first to show the cytotoxic activity of gambierone on mouse neuroblastoma cells while no cytotoxicity was observed when 44-MG was analysed at the same concentrations using the CBA-N2a. Analysis of the hydrophilic via LC-MS/MS confirmed production of gambierone in all isolates, ranging from 2.1 to 38.1 pg/cell, with 44-methylgambierone (44-MG) also produced by eight of the isolates, ranging from 0.3 to 42.9 pg/cell. No maitotoxin-1 was detected in any of the isolates. Classification of the G. honu strains according to the quantities of gambierone produced aligned with the classification of their cytotoxicity using the CBA-N2a. Finally, no maitotoxin-1 (MTX) was detected in any of the isolates. This study shows G. honu is widely distributed within the Pacific region with no significant intraspecific population structure present. This aligns with the view of microalgal populations as global metapopulations, however more in-depth assessment with other genetic markers could detect further structure. Toxicity diversity across 10 isolates assessed did not display any geographical patterns.

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.

Toxicology of Gambierdiscus spp. (Dinophyceae) from Tropical and Temperate Australian Waters.

Ciguatera Fish Poisoning (CFP) is a human illness caused by the consumption of marine fish contaminated with ciguatoxins (CTX) and possibly maitotoxins (MTX), produced by species from the benthic dinoflagellate genus Gambierdiscus. Here, we describe the identity and toxicology of Gambierdiscus spp. isolated from the tropical and temperate waters of eastern Australia. Based on newly cultured strains, we found that four Gambierdiscus species were present at the tropical location, including G. carpenteri, G. lapillus and two others which were not genetically identical to other currently described species within the genus, and may represent new species. Only G. carpenteri was identified from the temperate location. Using LC-MS/MS analysis we did not find any characterized microalgal CTXs (P-CTX-3B, P-CTX-3C, P-CTX-4A and P-CTX-4B) or MTX-1; however, putative maitotoxin-3 (MTX-3) was detected in all species except for the temperate population of G. carpenteri. Using the Ca2+ influx SH-SY5Y cell Fluorescent Imaging Plate Reader (FLIPR) bioassay we found CTX-like activity in extracts of the unidentified Gambierdiscus strains and trace level activity in strains of G. lapillus. While no detectable CTX-like activity was observed in tropical or temperate strains of G. carpenteri, all species showed strong maitotoxin-like activity. This study, which represents the most comprehensive analyses of the toxicology of Gambierdiscus strains isolated from Australia to date, suggests that CFP in this region may be caused by currently undescribed ciguatoxins and maitotoxins.

qPCR Assays for the Detection and Quantification of Multiple Paralytic Shellfish Toxin-Producing Species of Alexandrium.

Paralytic shellfish toxin producing dinoflagellates have negatively impacted the shellfish aquaculture industry worldwide, including in Australia and New Zealand. Morphologically identical cryptic species of dinoflagellates that may differ in toxicity, in particular, species of the former Alexandrium tamarense species complex, co-occur in Australia, as they do in multiple regions in Asia and Europe. To understand the dynamics and the ecological drivers of the growth of each species in the field, accurate quantification at the species level is crucial. We have developed the first quantitative polymerase chain reaction (qPCR) primers for A. australiense, and new primers targeting A. ostenfeldii, A. catenella, and A. pacificum. We showed that our new primers for A. pacificum are more specific than previously published primer pairs. These assays can be used to quantify planktonic cells and cysts in the water column and in sediment samples with limits of detection of 2 cells/L for the A. catenella and A. australiense assays, 2 cells/L and 1 cyst/mg sediment for the A. pacificum assay, and 1 cells/L for the A. ostenfeldii assay, and efficiencies of >90%. We utilized these assays to discriminate and quantify co-occurring A. catenella, A. pacificum, and A. australiense in samples from the east coast of Tasmania, Australia.

Alexandrium diversaporum sp. nov., a new non-saxitoxin producing species: Phylogeny, morphology and sxtA genes.

Species of the PST producing planktonic marine dinoflagellate genus Alexandrium have been intensively scrutinised, and it is therefore surprising that new taxa can still be found. Here we report a new species, Alexandrium diversaporum nov. sp., isolated from spherical cysts found at two sites in Tasmania, Australia. This species differs in its morphology from all previously reported Alexandrium species, possessing a unique combination of morphological features: the presence of 2 size classes of thecal pores on the cell surface, a medium cell size, the size and shape of the 6″, 1', 2⁗ and Sp plates, the lack of a ventral pore, a lack of anterior and posterior connecting pores, and a lack of chain formation. We determined the relationship of the two strains to other species of Alexandrium based on an alignment of concatenated SSU-ITS1, 5.8S, ITS2 and partial LSU ribosomal RNA sequences, and found A. diversaporum to be a sister group to Alexandrium leei with high support. A. leei shares several morphological features, including the relative size and shapes of the 6″, 1', 2⁗ and Sp plates and the fact that some strains of A. leei have two size classes of thecal pores. We examined A. diversaporum strains for saxitoxin production and found them to be non-toxic. The species lacked sequences for the domain A4 of sxtA, as has been previously found for non-saxitoxin producing species of Alexandrium.

MORPHOLOGY AND MOLECULAR PHYLOGENY OF ANKISTRODINIUM GEN. NOV. (DINOPHYCEAE), A NEW GENUS OF MARINE SAND-DWELLING DINOFLAGELLATES FORMERLY CLASSIFIED WITHIN AMPHIDINIUM(1).

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.

Are prorocentroid dinoflagellates monophyletic? A study of 25 species based on nuclear and mitochondrial genes.

The dinoflagellate sub-class Prorocentrophycidae has a distinct morphology, lacking the typical dinoflagellate cell structure of a clear cingulum and sulcus. It includes species that produce the toxin okadaic acid. Despite its uniqueness, the group has been found polyphyletic in some previous molecular phylogenetic studies. We have re-investigated the phylogeny of this sub-class by culturing and sequencing new strains, comparing sequences from three genes, the mitochondrial cytochrome c oxidase subunit 1 (cox 1) and the nuclear large and small subunit rRNA (LSU and SSU) encoding genes. We analyzed sequences from twenty-five named and two still undescribed species of Prorocentrophycidae. We used newly recognized features of the secondary structure to align regions of the LSU rRNA. The phylogeny based on cox 1 provided the most well-supported tree and showed strong support for the monophyly of prorocentroid dinoflagellates, while the LSU phylogeny was inconclusive. As in previous studies, phylogeny based on SSU shows the group to appear paraphyletic, however, support values were low. Two strongly supported sub-clades were consistently identified. Benthic and planktonic modes appear to have evolved on multiple occasions within both clades of Prorocentriphycidae. The capability to synthesize toxins appears to have arisen early in prorocentroid evolution and, in particular, okadaic acid synthesis is present in some, but not all, members of Clade 2. The D2a region of the LSU rRNA appears to have developed a deletion in three definable steps during prorocentroid evolution. While the phylogenies inferred from the three genes were not congruent, our results give reserved support to the monophyly of the group.

Ocean urea fertilization for carbon credits poses high ecological risks.

The proposed plan for enrichment of the Sulu Sea, Philippines, a region of rich marine biodiversity, with thousands of tonnes of urea in order to stimulate algal blooms and sequester carbon is flawed for multiple reasons. Urea is preferentially used as a nitrogen source by some cyanobacteria and dinoflagellates, many of which are neutrally or positively buoyant. Biological pumps to the deep sea are classically leaky, and the inefficient burial of new biomass makes the estimation of a net loss of carbon from the atmosphere questionable at best. The potential for growth of toxic dinoflagellates is also high, as many grow well on urea and some even increase their toxicity when grown on urea. Many toxic dinoflagellates form cysts which can settle to the sediment and germinate in subsequent years, forming new blooms even without further fertilization. If large-scale blooms do occur, it is likely that they will contribute to hypoxia in the bottom waters upon decomposition. Lastly, urea production requires fossil fuel usage, further limiting the potential for net carbon sequestration. The environmental and economic impacts are potentially great and need to be rigorously assessed.

Improving the analysis of dinoflagellate phylogeny based on rDNA.

Phylogenetic studies of dinoflagellates are often conducted using rDNA sequences. In analyses to date, the monophyly of some of the major lineages of dinoflagellates remain to be demonstrated. There are several reasons for this uncertainty, one of which may be the use of models of evolution that may not closely fit the data. We constructed and examined alignments of SSU and partial LSU rRNA along with a concatenated alignment of the two molecules. The alignments showed several characteristics that may confound phylogeny reconstruction: paired helix (stem) regions that contain non-independently evolving sites, high levels of compositional heterogeneity among some of the sequences, high levels of incompatibility (homoplasy), and rate heterogeneity among sites. Taking into account these confounding factors, we analysed the data and found that the Gonyaulacales, a well-supported clade, may be the most recently diverged order. Other supported orders were, in the analysis based on SSU, the Suessiales and the Dinophysiales; however, the Gymnodiniales and Prorocentrales appeared to be polyphyletic. The Peridiniales without Heterocapsa species appeared as a monophyletic group in the analysis based on LSU; however, the support was low. The concatenated alignment did not provide a better phylogenetic resolution than the single gene alignments.