Alexandrium Tamarense Species Complex Research Articles

First insights into the distribution and diversity of toxic dinoflagellate cysts in the surface sediments of Dakhla Bay (African Atlantic coast): relationships with environmental factors and mollusk intoxication events.

Dakhla Bay, situated on the African Atlantic coast, has witnessed sporadic harmful algal blooms (HABs) caused by toxic dinoflagellate species over the past two decades. In this study, we investigated the distribution, abundance, and diversity of dinoflagellate cysts, with a focus on potentially toxic species that develop in this ecosystem where such data are lacking. Sediment samples were collected in April 2018 through coring at 49 stations distributed across the bay. The highest abundance of dinoflagellate cysts was recorded at 304 cysts/g dry sediment, observed at the inner part of the bay, indicating that this area is the preferential zone for cyst accumulation. Pearson's tests revealed significant positive correlations (P < 0.05) between cyst abundance and the water content, organic matter, and fine fraction (< 63μm) of the sediment. Cyst morphotypes of potentially toxic dinoflagellate species known to produce saxitoxins, such as Alexandrium minutum, Alexandrium tamarense species complex, Gymnodinium catenatum, and yessotoxins, such as Lingulodinium polyedrum and Gonyaulax cf. spinifera, were identified in the sediment of Dakhla Bay. These findings were further supported by our long-term monitoring period (2005-2018), underscoring the presence of these HAB species in Dakhla Bay. During our survey, sporadic mollusk intoxication events were recorded at station PK25 for the grooved razor shell Solen marginatus and at station Boutelha for the oyster Crassostrea gigas. Paralytic shellfish toxin concentrations exceeded the sanitary threshold (80μg STX di-HCl eq/100g of shellfish meat) only twice, in December 2006 and January 2007 at station PK25. Contamination by amnesic shellfish toxins occurred sporadically but never reached the sanitary threshold of 20µg/g of shellfish meat. Lipophilic shellfish intoxication occurred multiple times in the two investigated areas. These observations suggest that the cysts of the identified HAB species germinated and inoculated the water column, resulting in the observed intoxication events. Relatively low levels of intoxication could be attributed to the moderate abundances of cysts, which may reduce the seeding capacity. This could be explained by the significant interaction of Dakhla Bay with the Atlantic Ocean, characterized by hydrological dynamics that impede the deposition and accumulation of cysts in the bay's sediments. This study reaffirms the importance of investigating dinoflagellate cysts in assessing the diversity of HAB species and evaluating associated sanitary risks.

The distribution of dinoflagellate cyst assemblages in recent sediments of the Oualidia Lagoon, Morocco, with a focus on toxic species

Harmful algal blooms (HABs) are becoming widely distributed and more frequent, threatening socioecosystems and human health. We determined species composition, abundance and spatial distribution of dinoflagellate cysts in the upper sediment of the Oualidia Lagoon located on the Atlantic coast of Morocco. Sediment samples were collected in 2017 at 51 stations, and environmental parameters were measured together with microphytoplankton abundance. Sediment characteristics including water percentage, organic matter content and grain size were determined. Fourteen dinoflagellate cyst morphotypes were identified, with Lingulodinium polyedrum (50%) and Gonyaulax spinifera (22%) dominating the assemblages. Total cyst densities ranged from 0 to 293 cysts g−1 dry sediment. Cyst densities were positively correlated with water content and organic matter content and increased with decreasing sediment grain size. We revealed the presence of three neurotoxic dinoflagellate species: Alexandrium minutum, the Alexandrium tamarense species complex, and Gymnodinium catenatum. Numerous cysts had accumulated in the sediment, and, because they are likely responsible for the initiation of HABs in Oualidia Lagoon, they should be monitored.

Resting cysts of Alexandrium catenella and A. pacificum (Dinophyceae) in the Bohai and Yellow Seas, China: Abundance, distribution and implications for toxic algal blooms

The Alexandrium tamarense species complex consists of 5 closely related species that are important bloom-forming dinoflagellates with a complex life cycle. The formation of resting cyst is a key strategy to resist harsh environmental conditions. In this study, the resting cysts of two major bloom-forming species of the A. tamarense species complex in China, A. catenella (Whedon & Kof.) Balech (previously A. fundyense, or A. tamarense species complex Group I) and A. pacificum Litaker (A. tamarense species complex Group IV), were studied in surface sediment collected from the Bohai Sea (BS) and Yellow Sea (YS) during two cruises conducted in 2012 and 2015. Cyst abundance of the A. tamarense species complex was first quantified by the primuline-staining method, and cysts of the two species were subsequently determined using two real-time quantitative PCR (qPCR) assays. Results showed that resting cysts of the A. tamarense species complex were more abundant in the YS than the BS (mean of 480 and 33 cysts g dry weight, DW−1 of sediment, respectively). Cysts were mainly found in the central portion of the northern YS, the area SE (southeast) of the Shandong peninsula, and the area near the Subei Shoal in the southern YS, where surface sediment had a high percentage of clay and silt (particle size < 63 μm) content. The maximum cyst abundance recorded was 3090 cysts g DW−1 of sediment in 2012 and 3448 cysts g DW−1 in 2015, respectively. Cysts were mainly composed of A. catenella in the YS and the BS, while those of A. pacificum were only detected occasionally at some sampling sites in the YS. Highly abundant resting cysts in surface sediment of the YS may serve as “seed banks” for recurrent toxic blooms of A. catenella and the associated shellfish contamination by paralytic shellfish toxins in the YS.

The Cost of Toxicity in Microalgae: Direct Evidence From the Dinoflagellate Alexandrium.

Empirical evidence of the cost of producing toxic compounds in harmful microalgae is completely lacking. Yet costs are often assumed to be high, implying substantial ecological benefits with adaptive significance exist. To study potential fitness costs of toxin production, 16 strains including three species of the former Alexandrium tamarense species complex were grown under both carbon limitation and unlimited conditions. Growth rates, levels of intracellular paralytic shellfish toxins (PSTs), and effects of lytic compounds were measured to provide trade-off curves of toxicity for both PST and lytic toxicity under high light (300 μmol photons m−2 s−1) and under low light (i.e., carbon limited; 20 μmol photons m−2 s−1). Fitness costs in terms of reduced growth rates with increasing PST content were only evident under unlimited conditions, but not under carbon limitation, in which case PST production was positively correlated with growth. The cost of production of lytic compounds was detected both under carbon limitation and unlimited conditions, but only in strains producing PST. The results may direct future research in understanding the evolutionary role and ecological function of algal toxins. The intrinsic growth rate costs should be accounted for in relation to quantifying benefits such as grazer avoidance or toxin-mediated prey capture in natural food web settings.

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.

Morphological, molecular, and toxin analysis of field populations of Alexandrium genus from the Argentine Sea

In the Argentine Sea, blooms of toxigenic dinoflagellates of the Alexandrium tamarense species complex have led to fish and bird mortalities and human deaths as a consequence of paralytic shellfish poisoning (PSP). Yet little is known about the occurrence of other toxigenic species of the genus Alexandrium, or of their toxin composition beyond coastal waters. The distribution of Alexandrium species and related toxins in the Argentine Sea was determined by sampling surface waters on an oceanographic expedition during austral spring from ~39°S to 48°S. Light microscope and SEM analysis for species identification and enumeration was supplemented by confirmatory PCR analysis from field samples. The most frequent Alexandrium taxon identified by microscopy corresponded to the classical description of A.tamarense. Only weak signals of Group I from the A.tamarense species complex were detected by PCR of bulk field samples, but phylogenetic reconstruction of rDNA sequences from single cells from one station assigned them to ribotype Group I (Alexandrium catenella). PCR probes for Alexandrium minutum and Alexandrium ostenfeldii yielded a positive signal, although A.minutum morphology did not completely match the classical description. Analysis of PSP toxin composition of plankton samples revealed toxin profiles dominated by gonyautoxins (GTX1/4). The main toxic cyclic imine detected was 13-desMe-spirolide C and this supported the association with A.ostenfeldii in the field. This study represents the first integrated molecular, morphological and toxinological analysis of field populations of the genus Alexandrium in the Argentine Sea.

Bioactive compounds of marine dinoflagellate isolates from western Greenland and their phylogenetic association within the genus Alexandrium

The diversity and biogeography of populations of the toxigenic marine dinoflagellate genus Alexandrium, a major global cause of paralytic shellfish poisoning (PSP), are represented by only a few studies based upon a low number of cultured isolates and remain poorly described in Arctic and sub-Arctic waters. Multiple clonal isolates (n=22) of the Alexandrium tamarense species complex, and a single isolate of A. tamutum, were collected from the water column while on board an oceanographic expedition to the west coast of Greenland. After culturing of these isolates under controlled conditions, their phylogenetic affinities within the genus Alexandrium were characterized by sequence analysis of nuclear large sub-unit (LSU) rDNA. Based upon morphological and molecular genetic criteria, all isolates of the A. tamarense species complex were consistent with membership in the Group I ribotype (previously known as the North American ribotype). Phenotypic signatures were also analyzed based upon their respective profiles of paralytic shellfish toxins (PST) and allelochemical interactions against a target cryptophyte Rhodomonas, as determined by lytic potency. All isolates conforming to the A. tamarense Group I produced PST, but no toxins were detected in A. tamutum P2E2. Unusually, only carbamoyl toxins were produced among the A. tamarense Group I isolates from Greenland; sulfocarbamoyl derivatives, generally present in A. tamarense population from other locations, including the Arctic, North Pacific and North Atlantic, were absent from all isolates. Allelochemical activity, causing cell lysis of Rhodomonas, but generally being unrelated to cellular PST, was expressed by all A. tamarense isolates and also by A. tamutum, but varied widely in potency. Comparison of the genotypic (rDNA) and phenotypic (PST profile, allelochemical activity) characteristics of Greenland isolates with those of other Arctic populations reveals a complex pattern of intra-specific diversity. Estimation of diversity relationships is problematic because of the distinct patterns of divergence and lack of evidence of linkage among the alternative biomarkers and morphology. Nevertheless, such studies are necessary as the basis for constructing hindcasting scenarios and predicting changes in Alexandrium species distribution in the Arctic from the regional to the global scale.

Variability in the allelopathic action of the Alexandrium tamarense species complex along the coast of China

Members of the Alexandrium tamarense species complex are some of the most important toxigenic dinoflagellates and are also widely distributed along the coast of China. A. tamarense and another well-known toxic raphidophycean alga, Chattonella marina, are usually found in the same sea areas, sometimes at the same place and time. In this study, under laboratory co-culture conditions, we found that most of the 15 A. tamarense strains from the South China Sea and the East China Sea had significant inhibition on the growth of C. marina, while most of the A. tamarense strains were inhibited to various extents by C. marina. These results suggest that there is a complex reciprocal inhibitory effect between A. tamarense and C. marina, which might be caused by their allelopathic potency, the intrinsic growth rate of the algal species and the sensitivity of the target alga to the allelochemicals, etc. However, no strict correlations were observed between the allelopathic actions and the geographical distribution. The allelopathic potency observed in co-culture did not correlate with the hemolytic activity of the extracellular toxins obtained by the solvent extraction method (p>0.05) but did correlate with the hemolytic activity obtained by the dialysis method (p<0.01). These results indicate that various strains of A. tamarense could produce diverse hemolytic substances with different compositions and properties, which might be responsible for the variability in A. tamarense allelopathic action.

Molecular phylogeny and PSP toxin profile of the Alexandrium tamarense species complex along the coast of China

To explore the genetic diversity and paralytic shellfish poisoning (PSP) toxin profile of the Alexandrium tamarense species complex along the coast of China, 67 strains of A. tamarense from the China Sea were collected and genetic diversity were analyzed based on the rDNA sequences. In addition, PSP toxin compositions and contents were detected by HPLC. According to the 5.8S rDNA and ITS, and LSU rDNA D1–D2 sequence, A. tamarense in the China Sea comprises at least Group IV and Group I ribotypes. In these Chinese strains, the toxins with the highest concentration in the profile were C1/2, gonyautoxins 1/4 (GTX1/4) and neosaxitoxin (NEO). However, the toxin profiles were atypical and C1/2 toxins were not detected in some strains. No strict correlation was observed between the PSP toxins profile and the geographical distribution.

Formal Revision of the Alexandrium tamarense Species Complex (Dinophyceae) Taxonomy: The Introduction of Five Species with Emphasis on Molecular-based (rDNA) Classification

The Alexandrium tamarense species complex is one of the most studied marine dinoflagellate groups due to its ecological, toxicological and economic importance. Several members of this complex produce saxitoxin and its congeners - potent neurotoxins that cause paralytic shellfish poisoning. Isolates from this complex are assigned to A. tamarense, A. fundyense, or A. catenella based on two main morphological characters: the ability to form chains and the presence/absence of a ventral pore between Plates 1' and 4'. However, studies have shown that these characters are not consistent and/or distinctive. Further, phylogenies based on multiple regions in the rDNA operon indicate that the sequences from morphologically indistinguishable isolates partition into five clades. These clades were initially named based on their presumed geographic distribution, but recently were renamed as Groups I-V following the discovery of sympatry among some groups. In this study we present data on morphology, ITS/5.8S genetic distances, ITS2 compensatory base changes, mating incompatibilities, toxicity, the sxtA toxin synthesis gene, and rDNA phylogenies. All results were consistent with each group representing a distinct cryptic species. Accordingly, the groups were assigned species names as follows: Group I, A. fundyense; Group II, A. mediterraneum; Group III, A. tamarense; Group IV, A. pacificum; Group V, A. australiense.

(2302) Proposal to reject the name Gonyaulax catenella (Alexandrium catenella) (Dinophyceae).

TAXONVolume 63, Issue 4 p. 932-933 Proposals to Conserve or Reject NamesFree Access (2302) Proposal to reject the name Gonyaulax catenella (Alexandrium catenella) (Dinophyceae) Uwe John, Corresponding Author Uwe John uwe.john@awi.de Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, Bremerhaven, 27570 GermanySearch for more papers by this authorWayne Litaker, Wayne Litaker National Oceanic and Atmospheric Administration, National Ocean Service, National Centers for Coastal Oceans Science, Center for Fisheries and Habitat Research, 101 Pivers Island Road, Beaufort, North Carolina, 28516 U.S.A.Search for more papers by this authorMarina Montresor, Marina Montresor Stazione Zoologica Anton Dohrn, Villa Comunale, Napoli, 80121 ItalySearch for more papers by this authorShauna Murray, Shauna Murray Plant Functional Biology and Climate Change Cluster, University of Technology, Sydney, P.O. Box 123 Broadway, New South Wales, 2007 AustraliaSearch for more papers by this authorMichael L. Brosnahan, Michael L. Brosnahan Woods Hole Oceanographic Institution, MS # 32, 266 Woods Hole Road, Woods Hole, Massachusetts, 02543 U.S.A.Search for more papers by this authorDonald M. Anderson, Donald M. Anderson Woods Hole Oceanographic Institution, MS # 32, 266 Woods Hole Road, Woods Hole, Massachusetts, 02543 U.S.A.Search for more papers by this author Uwe John, Corresponding Author Uwe John uwe.john@awi.de Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, Bremerhaven, 27570 GermanySearch for more papers by this authorWayne Litaker, Wayne Litaker National Oceanic and Atmospheric Administration, National Ocean Service, National Centers for Coastal Oceans Science, Center for Fisheries and Habitat Research, 101 Pivers Island Road, Beaufort, North Carolina, 28516 U.S.A.Search for more papers by this authorMarina Montresor, Marina Montresor Stazione Zoologica Anton Dohrn, Villa Comunale, Napoli, 80121 ItalySearch for more papers by this authorShauna Murray, Shauna Murray Plant Functional Biology and Climate Change Cluster, University of Technology, Sydney, P.O. Box 123 Broadway, New South Wales, 2007 AustraliaSearch for more papers by this authorMichael L. Brosnahan, Michael L. Brosnahan Woods Hole Oceanographic Institution, MS # 32, 266 Woods Hole Road, Woods Hole, Massachusetts, 02543 U.S.A.Search for more papers by this authorDonald M. Anderson, Donald M. Anderson Woods Hole Oceanographic Institution, MS # 32, 266 Woods Hole Road, Woods Hole, Massachusetts, 02543 U.S.A.Search for more papers by this author First published: 01 August 2014 https://doi.org/10.12705/634.21Citations: 25AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume63, Issue4August 2014Pages 932-933 RelatedInformation

Influences of sedimentation and hydrodynamics on the spatial distribution of Alexandrium catenella/tamarense resting cysts in a shellfish farming lagoon impacted by toxic blooms

Since resting cysts are a potential seeding source for blooms, the presence of these cysts in sediments is a marker of an established population for a number of harmful algal species. The spatial patterns of cyst density in relation to sediment characteristics and hydrodynamics are still largely misunderstood. This study investigated the spatial distribution of resting cysts belonging to the Alexandrium tamarense species complex (Dinophyceae) in sediments of a Mediterranean coastal lagoon (Thau Lagoon, France). This lagoon, hosting shellfish farming, is regularly impacted by toxic Alexandrium catenella blooms. The average cyst density across the whole lagoon was rather low, <20cystsg−1 of dry sediment (DS). However, densities varied widely among sampled stations, with the highest density (∼440cystsg−1 DS) recorded in a shallow cove named Crique-de-l’Angle, which is the only area where dense blooms of A. catenella and A. tamarense have been recorded in the years preceding this survey. An analysis using spatial autoregressive models demonstrated that cyst densities were highly spatially autocorrelated (indicating that close stations tended to have more similar cyst densities) with accumulation sites. With respect to sediment characteristics (5 granulometric fractions <2mm and biochemical components), the highest densities were found in silty sediments containing high proportions of water and organic matter. Nevertheless, the linkage between cyst density and sediment structure was not always verified; this reflected the influence of hydrodynamics on the sedimentation of cysts and sediment particles, and on the dispersal of cysts away from the bloom area by wind-induced currents, suggesting that hydrodynamics was responsible for the spatially autocorrelated distribution of cyst densities.

High cyst concentrations of the potentially toxic dinoflagellate Alexandrium tamarense species complex in Bedford Basin, Halifax, Nova Scotia, Canada

We report a large cyst bed of the potentially toxic and bloom-forming dinoflagellate Alexandrium tamarense species complex in bottom sediments from the port of Halifax, Nova Scotia, Canada. The average cyst concentrations of that species ranged from 4033±2647 to 220872±148086cystsg−1 of dry sediments and the highest concentrations were found near ship terminals in Bedford Basin. Although this species is endemic to this region, our work strongly suggests that some of the cysts of A. tamarense species complex found in the port of Halifax were introduced through discharged ballast water and sediments.

Reactive oxygen species are linked to the toxicity of the dinoflagellate Alexandrium spp. to protists

Short-term experiments were conducted to examine the response of the ciliate Tia- rina fusus and the heterotrophic dinoflagellate Polykrikos kofoidii to 3 strains in the Alexandrium tamarense species complex, each with a different paralytic shellfish toxin (PST) content. Both pro- tist species fed on all 3 Alexandrium strains, but significant mortality occurred within 24 h of initial exposure to high densities of each dinoflagellate isolate. Protist mortality was not related, how- ever, to the PST content of the Alexandrium strains, indicating a different mechanism of toxicity. Exposure of T. fusus to cell-free culture filtrates or cell extracts did not cause significant ciliate mortality. In contrast, significant mortality occurred when ciliates were separated physically from a live Alexandrium sp. culture by a 10 µm nylon mesh, suggesting that the toxicity is dependent upon the viability of the Alexandrium spp. cells but does not require physical contact or ingestion. Addition of antioxidant compounds significantly increased the survival of both protist species when exposed to Alexandrium, suggesting that reactive oxygen species and/or the secondary compounds produced by ROS-induced lipid peroxidation are involved in the toxicity of Alexan- drium spp. to ciliates and heterotrophic dinoflagellates. This mechanism of toxicity is previously unknown for Alexandrium spp. and may play an important role in bloom dynamics and toxin transfer within the food web.

Intraregional variation among Alexandrium catenella (Dinophyceae) strains from southern Chile: Morphological, toxicological and genetic diversity

Morphological, toxicological and phylogenetic analyses, using the partial LSU gene and internal spacer (ITS) regions of the rDNA gene, were combined to evaluate the intraregional diversity of Alexandrium catenella occurring along the southern coast of Chile. Twenty-two strains isolated from different localities along the wide area of distribution of the species (from 42°S to 55°S) were examined by these three approaches. Morphologically, although the strains showed diagnostic characters according to the species definition, variations in these traits within and between strains were also observed. The absence of an apical or posterior attachment pore, for instance, was observed mainly in old isolates. Indirect connection between the apical and 1′ plates, traits normally seen in other species of the same genus, was also noted in some strains. However, the lack of a ventral pore on the 1′ plate was one of the most distinctive characteristics present in all the Chilean strains. Toxicologically, the Chilean strains were characterized by the dominance of N-sulfocarbamate (C1,2) and gonyautoxins (GTX1–4), but also by the scarcity or absence of saxitoxin. Considering the dominance of these toxins in each strain, at least two distinctive toxin patterns were distinguished. Through rDNA sequence analysis, the Chilean strains were segregated as part of Clade I (North American) of the Alexandrium tamarense species complex. Nevertheless, significant genetic diversity was also observed among the Chilean strains, especially using ITS sequences. Through these three approaches, Chilean strains of A. catenella showed significant intraregional variability, which is appropriate for a native species. However, the distribution of its genetic diversity seems to be inconsistent with the apparent northward expansion observed along the west coast of South America.

Detecting copepod grazing on low-concentration populations of Alexandrium fundyense using PCR identification of ingested prey

Zooplankton grazing is often a significant loss term for phytoplankton populations, including harmful algae, impacting the development and decline of blooms. However, detecting and quantifying predation on phytoplankton is often challenging, particularly during early bloom stages when phytoplankton cell concentrations are low. In this study, we used polymerase chain reaction (PCR) to detect ingestion of toxic dinoflagellates of the Alexandrium tamarense species complex by two copepods, Acartia hudsonica (laboratory population) and Calanus finmarchicus (field population). Recent ingestion of Alexandrium fundyense cells was indicated by positive amplification of an LSU rDNA fragment specific to A. fundyense from whole copepod extracts. In laboratory experiments, A. fundyense DNA was detectable for 2–4 h post-ingestion in A. hudsonica fed A. fundyense, but not detected in animals fed other phytoplankton, or starved. In field samples, ingestion of A. fundyense by C. finmarchicus was confirmed by PCR, including at four stations where the A. fundyense concentration was ≤14 cells L−1. At these low prey concentrations, ingestion rates on A. fundyense may have been as low as 1 cell copepod−1 day−1. Nevertheless, simulations of A. fundyense population growth suggest that a few predators L−1 have the potential to curb the early development of a slow-growing bloom, even if ingestion rates are extremely low. Low predation rates can still have a large impact when prey populations are small.

IDENTIFICATION OF THE TOXIC ALGA ALEXANDRIUM TAMIYAVANICHI (DINOPHYCEAE) FROM NORTHEASTERN BRAZIL: A COMBINED MORPHOLOGICAL AND rDNA SEQUENCE (PARTIAL LSU AND ITS) APPROACH1

Morphological and phylogenetic analyses and sequencing of the partial LSU gene and internal transcribed spacer (ITS) regions of the rDNA gene were combined to study toxic strains of Alexandrium tamiyavanichi Balech collected from northeastern Brazilian coastal waters. All specimens were identified with A. tamiyavanichi mainly based on the shape of the anterior sulcal plate (S.a.), which was the most conservative character. Among the specimens studied, 8% did not conform to the morphological description of A. tamiyavanichi. The occurrence of transitory states between A. tamiyavanichi and Alexandrium cohorticula (Balech) Balech tended to confirm that both species are conspecific. The posterior sulcal plate (S.p.) was not a good taxonomic character as variability in its shape did not allow a clear assignment of specimens from the same clonal isolate to either morphologically defined species. Phylogenetic analyses confirmed the overall validity of morphological characters to delineate the evolutionary relationships among the clades of Alexandrium species analyzed here, indicating that A. tamiyavanchi is a valid species according to the morphological species concept. This analysis showed that the Brazilian strains form a monophyletic clade with Asiatic strains of A. tamiyavanichi, but with enough genetic distance to argue for long‐term separation and isolation of locally established populations, extending the known biogeographic range of this species. On the higher phylogenetic level, our data also indicate that Alexandrium tropicale Balech forms part of the clade otherwise consisting of the morphologically defined freterculus group, and this A. tamiyavanichi/tropicale/fraterculus (Balech) Balech clade (including species from the formerly termed tropical Asian [TROP] clade) may be considered as a sister group of the Alexandrium tamarense species complex.

Outbreeding lethality between toxic Group I and nontoxic Group III Alexandrium tamarense spp. isolates: Predominance of heterotypic encystment and implications for mating interactions and biogeography.

We report the zygotic encystment of geographically dispersed isolates in the dinoflagellate species complex Alexandrium tamarense, in particular, successful mating of toxic Group I and nontoxic Group III isolates. However, hypnozygotes produced in Group I/III co-cultures complete no more than three divisions after germinating. Previous reports have suggested a mate recognition mechanism whereby hypnozygotes produced in co-cultures could arise from either homotypic (inbred) or heterotypic (outbred) gamete pairs. To determine the extent to which each occurs, a nested PCR assay was developed to determine parentage of individual hypnozygotes. The vast majority of hypnozygotes from pairwise Group I/III co-cultures were outbred, so that inviability was a result of hybridization, not inbreeding. These findings support the assertion that complete speciation underlies the phylogenetic structure of the Alexandrium tamarense species complex. Additionally, the ribosomal DNA (rDNA) copy numbers of both hybrid and single ribotype hypnozygotes were reduced substantially from those of haploid motile cells. The destruction of rDNA loci may be crucial for the successful mating of genetically distant conjugants and appears integral to the process of encystment. The inviability of Group I/III hybrids is important for public health because the presence of hybrid cysts may indicate ongoing displacement of a nontoxic population by a toxic one (or vice versa). Hybrid inviability also suggests a bloom control strategy whereby persistent, toxic Group I blooms could be mitigated by introduction of nontoxic Group III cells. The potential for hybridization in nature was investigated by applying the nested PCR assay to hypnozygotes from Belfast Lough, Northern Ireland, a region where Group I and III populations co-occur. Two hybrid cysts were identified in 14 successful assays, demonstrating that Group I and III populations do interbreed in that region. However, an analysis of mating data collected over an 18-year period indicated a leaky pre-mating barrier between ribosomal species (including Groups I and III). Whether the observed selectivity inhibits hybridization in nature is dependent on its mechanism. If the point of selectivity is the induction of gametogenesis, dissimilar ribotypes could interbreed freely, promoting displacement in cases where hybridization is lethal. If instead, selectivity occurs during the adhesion of gamete pairs, it could enable stable coexistence of A. tamarense species. In either case, hybrid inviability may impose a significant obstacle to range expansion. The nested PCR assay developed here is a valuable tool for investigation of interspecies hybridization and its consequences for the global biogeography of these important organisms.

Dormancy and germination features in resting cysts of Alexandrium tamarense species complex (Dinophyceae) can facilitate bloom formation in a shallow lagoon (Thau, southern France)

This study investigated the dormancy length, germination patterns and cyst progeny fate of Alexandrium catenella and A. tamarense found in Thau lagoon, France. In laboratory-produced cysts, the dormancy period was estimated to be less than 2 weeks. A vernalization period was not required prior to germination. However, after 1 month of dark-storage at 6°C, excystment was obtained 2–3 days from re-exposure to favourable conditions thereby suggesting that germination was synchronized. Germination patterns and germling cell viability in laboratory-produced cysts were similar to those exhibited in natural cysts. The following conditions optimized germination: temperature between 14 and 26°C, salinity between 30 and 38 psu and light exposure for >1 h at 100 μmoles photons m−2 s−1. Similar conditions prevailed at the commencement of and during Alexandrium blooms in Thau lagoon, suggesting that cysts may germinate shortly after being recruited from sediment re-suspensions. Accordingly, cyst bank germinations contribute to the initial seeding of blooms, and subsequently germinations of newly formed cysts can help to maintain blooms. In cyst banks, the high germination capabilities seem, however, to be counter-balanced by the low viability of cyst progeny, as estimated from the realized seeding ratio. This ratio is proposed as a means of quantifying the potential for cyst banks to regenerate planktonic populations.

Comparative studies on morphology, ITS sequence and protein profile of Alexandrium tamarense and A. catenella isolated from the China Sea

The Alexandrium tamarense species complex is a closely related cosmopolitan toxigenic group of morphology-based species, including A. tamarense, A. catenella and A. fundyense. This study investigated the morphology, internal transcribed spacer (ITS) sequence and protein profile of A. tamarense and A. catenella grown in the same culture conditions using a combination of scanning electronic microscope (SEM), molecular and proteomic approaches. The results showed that all Alexandrium strains had the plate formula of Po, 4′, 6″, 6C, 8S, 5″′, 2″″. The ventral pore, a key conventional morphological feature to discriminate A. tamarense and A. catenella, was usually present in the first apical plate of ten A. tamarense strains, however, it was found to be absent in some cells of one Alexandrium strain, ATGX01. A. tamarense and A. catenella shared an identical ITS sequence with a minor variation at intraspecific level. Protein profiles of A. catenella DH01 and A. tamarense DH01, isolated from the same region of the East China Sea, showed no significant difference, the similarity of protein profiles of the two species reached 99% with a few proteins unique to one or the other. The present results suggest that the ventral pore is not a consistent morphological feature in the Alexandrium genus, and that A. tamarense and A. catenella are conspecific and should be redesignated to one species.