Heterocapsa Species Research Articles

Characterization and inter-strain variability in ichthyotoxicity of Heterocapsa ovata (Peridiniales, Dinophyceae) from temperate waters of South Australia

IntroductionHarmful algal bloom (HAB)-forming species and populations exhibit substantial intraspecific functional trait variation, which can confer eco-evolutionary advantages. Phenotypic variability among populations can buffer the immediate detrimental effects of environmental fluctuations, with more diverse populations expected to survive changing conditions more efficiently than their uniform counterparts.MethodsIn February 2014, a mixed fish-killing dinoflagellate bloom occurred in the temperate waters of Coffin Bay, South Australia, causing the death of oysters and fish in the area. The bloom was dominated by Karenia mikimotoi and a cryptic species of Heterocapsa. Twenty-one monoclonal Heterocapsa isolates were established from the site and identified as H. ovata using microscopy and universal ribosomal markers (ITS/5.8S and LSU D1/D3 rDNA regions; SSU and cob were used for amplicon sequencing). These isolates were tested for ichthyotoxicity using a bioassay based on cells from the gills of rainbow trout (Oncorhynchus mykiss). Culture fraction preparations (whole cells, supernatant, and lysed cells) were analyzed to determine ichthyotoxicity levels.ResultsThe highest ichthyotoxicity was observed in lysed cells, with surprisingly high inter-strain variability. This suggests that different strains of H. ovata have varying levels of toxicity.DiscussionResults from this study expand our understanding of the adaptive strategies of HAB species and enable predictions of future population dynamics under changing climatic conditions. The substantial phenotypic variability among H. ovatastrains highlights the potential for diverse responses to environmental stressors, underscoring the importance of considering intraspecific variation in ecological and evolutionary studies of HABs.

Isolation, identification and toxicity of three strains of Heterocapsa (Dinophyceae) in a harmful event in Fujian, China

Heterocapsa is a genus of dinoflagellates including species that can form harmful algae blooms (HABs) and cause deleterious ecological effects. To date two species ( H circularisquama and H bohaiensis) are known to be toxic. Here we isolated three strains of Heterocapsa from a multi-species dinoflagellate bloom in the aquaculture area of Fujian, China in June, 2019 that caused mass mortality of farmed abalone. Morphological analysis using light, transmission electron and scanning electron microscopy along with phylogenetic analyses with small (SSU) and large (LSU) subunit rRNA and internal transcribed spacer (ITS) gene sequences showed that these strains were H. cf. niei H. horiguchii, and H. cf. pygmaea, respectively. Furthermore, rabbit erythrocyte assay revealed hemolytic activity in all three strains in cell density dependent fashion, and only in the presence of light. In addition, the strains caused significant mortality of Artemia salina, and the toxicity was also cell density dependent. The Heterocapsa cultures and toxicity information obtained in this study expanded our knowledge of toxic species of Heterocapsa, and will facilitate further investigating the mechanism of their toxicity and developing monitoring tools for their blooms in the future.

Six marine thecate Heterocapsa (Dinophyceae) from Malaysia, including the description of three novel species and their cytotoxicity potential

Thirty-four strains of Heterocapsa were established from Malaysian waters and their morphologies were examined by light, scanning, and transmission electron microscopy. Three species, H. bohaiensis, H. huensis, and H. rotundata, and three new species, H. borneoensis sp. nov., H. limii sp. nov., and H. iwatakii sp. nov. were described in this study. The three species were differentiated morphologically by unique characteristics of cell size, shape, displacement of the cingulum, shape and position of nucleus, the number and position of pyrenoids, and body scale ultrastructure. The species delimitations were robustly supported by the molecular data. A light-microscopy-based key to species of Heterocapsa is established, with two major groups, i.e., species with a single pyrenoid, and species with multiple pyrenoids. Bioassays were conducted by exposing Artemia nauplii to Heterocapsa densities of 1–5 × 105 cells mL−1, and treatments exposed to H. borneoensis showed naupliar mortality, while no naupliar death was observed in the treatments exposed to cells of H. bohaiensis, H. huensis, H. limii, and H. iwatakii. Naupliar death was observed during the initial 24 h for both tested H. borneoensis strains, and mortality rates increased up to 50% after 72-h exposure. This study documented for the first time the diversity and cytotoxic potency of Heterocapsa species from Malaysian waters.

Diversity of <i>Heterocapsa</i> (Dinophyceae) and the algal bloom event in the mariculture areas of Johor Strait, Malaysia

In November 2020, a high biomass multi-species algal bloom caused heavy water discoloration in the fish and mussel farm areas of the Johor Strait, Malaysia. A total of 19 microalgal taxa were identified from the plankton samples collected during the bloom event. Eleven genera were diatoms, and eight genera were dinophytes. The microalgal composition was dominated by the diatom Guinardia sp., with an average cell density of 1.7×106 cells L−1, making up 65–80% of the phytoplankton composition. Concomitantly, high densities of the dinophytes Heterocapsa minima (3.8–5.3×105 cells L−1) and Karlodinium spp. (3.5–6.6×103 cells L−1) were found. This is the first record of the occurrence of H. minima in Malaysian waters. Detailed morphological observations of H. minima based on scanning electron microscopy are presented in this study. To have a better insight into the Heterocapsa species assemblages in the Johor Strait, the diversity of Heterocapsa species assemblages along the strait was investigated based on a metabarcoding approach. Environmental DNA collected between 2018 and 2019 was used for high throughput amplicon sequencing targeting the small subunit (SSU) ribosomal RNA gene marker. The metabarcoding analysis detected three rare Heterocapsa species in the waters, H. niei, H. rotundata, and H. steinii. The results showed that Heterocapsa species assemblages varied temporally across the strait, with higher species diversity and amplicon sequence variant (ASV) read abundances detected in the Eastern Johor Strait. Although no fish/shellfish kills were sighted during the 2020 bloom event, the presence of harmful microalgal species, such as Heterocapsa minima and Karlodinium spp., urged the need for a comprehensive HAB monitoring program in the Strait to safeguard the aquaculture industry in the areas.

Thecal tabulation, body scale morphology and phylogeny of Heterocapsa philippinensis sp. nov. (Peridiniales, Dinophyceae) from the Philippines

The thecal tabulation and body scale structure of the marine armoured dinoflagellate Heterocapsa, isolated from Philippines, were examined using LM, SEM and TEM, and its phylogenetic position was inferred from ITS and LSU rDNA sequences. Cells were ovoid and the plate tabulation (Po, cp, X, 5′, 3a, 7′′, 6c, 5s, 5′′′, 2′′′′) was consistent with most Heterocapsa species. The second anterior intercalary plate (2a) had a circular pattern with a thick marginal border free of pores. The nucleus was longitudinally elongated and curved, and located at the dorsal side of the cell. Discoid lobes of brownish chloroplast were peripherally distributed, and a pyrenoid was positioned at the centre. The triradiate body scales, measuring 250–300 nm in diameter, consisted of a roundish basal plate with six radiating ridges, nine peripheral uprights/spines, and three radiating spines. These components were identical to those of H. pseudotriquetra and H. steinii, except for the roundish outline of basal plate. Molecular phylogeny showed that the species clustered with H. pseudotriquetra and H. steinii. This species was differentiated from all other Heterocapsa species in the sausage-shaped nucleus and circular pattern on the 2a plate. This study proposed a novel species Heterocapsa philippinensis sp. nov. for the isolate.

Heterocapsa busanensis sp. nov. (Peridiniales, Dinophyceae): A new marine thecate dinoflagellate from Korean coastal waters

During daily monitoring in Yongho Bay off Busan, Korea in 2019, an isolate of the dinoflagellate genus Heterocapsa was established in clonal culture. Light and electron microscopic examination revealed that the isolate was ellipsoid in shape, exhibiting a thecal plate arrangement (Po, cp, X, 5′, 3a, 7″, 6c, 5s, 5‴, 2ʹʹʹʹ) consistent with most other Heterocapsa species. A large, elongated nucleus was positioned on the left side of the cell, a single reticulate chloroplast was located peripherally, and a single, starch-sheathed, spherical pyrenoid was present in the episome or near the cingulum. Morphologically, the isolate most closely resembles H. circularisquama and H. illdefina. Transmission electron microscopic examination of whole mounts revealed that the isolate had two body scale types, one of which was a complex, three-dimensional, fine structure distinct from other Heterocapsa species, whereas the other simpler type was structurally similar to the scales of H. horiguchii. Molecular phylogeny based on rRNA sequences revealed that the isolate was distantly related to morphologically similar species, but formed a sister lineage to H. horiguchii, a species characterized by a similar body scale morphology. Based on morphological, ultrastructural, and molecular data, we proposed it as a new species, Heterocapsa busanensis sp. nov.

Morphological and molecular characterization of Heterocapsa claromecoensis sp. nov. (Peridiniales, Dinophyceae) from Buenos Aires coastal waters (Argentina)

ABSTRACT A new species of the marine dinophyte genus Heterocapsa Stein is described. Two clonal strains originating from Argentinean coastal waters were examined with light and electron microscopy and LSU and ITS rDNA sequence data were obtained. Heterocapsa claromecoensis sp. nov. is described as a distinctive species with a conical epitheca, a similar sized and rounded hypotheca, a single reticulate chloroplast situated in the periphery of the cell, and a large pyrenoid in the episome, above an ellipsoidal to rounded nucleus in the hyposome. The plate tabulation pattern of Po (pore plate), cp (cover plate), X (X-plate or canal plate), 5ꞌ, 3a, 7ꞌꞌ, 6c, 5s, 5ꞌꞌꞌ, 2ꞌꞌꞌꞌ was as for most other species of Heterocapsa. H. claromecoensis sp. nov. differs from all other species of Heterocapsa by the microarchitecture of two different types of organic body scales, which uniquely were different in height. Phylogenetic trees based on LSU and ITS rDNA sequences placed H. claromecoensis in a separate branch, with a sequence assigned to H. orientalis being the closest match based on LSU rDNA.

Heterocapsa bohaiensis sp. nov. (Peridiniales: Dinophyceae): a novel marine dinoflagellate from the Liaodong Bay of Bohai Sea, China

A small armed dinoflagellate bloomed in the aquaculture ponds off the coast of Liaodong Bay, Bohai Sea of China, resulting in heavy mortalities of the cultured prawns (Penaeus japonicus) and larvae of Chinese mitten handed crabs (Eriocheir sinensis). The bloom-forming species was successfully isolated, and cellular morphology of the specimen was consequently investigated through light, fluorescent and electron microscopy. The small ((14.4±1.6) μm in length) ellipsoid cells show typical Heterocapsa thecal plate arrangement (Po, cp, 5′, 3a, 7′′, 6c, 5s, 5′′′, 2′′′′). The episome is evidently bigger than the hyposome. One to three spherical pyrenoids are located above or beside the large elongated nucleus. The body scale is characterized by a triangle basal plate with one central upright and nine peripheral spines. Above all, Heterocapsa bohaiensis could be distinguished from other Heterocapsa species by the combination of the cell size, morphology, cellular structure and body scale. Sequence analyses of both ITS and LSU regions reveal the significant genetic divergence between H. bohaiensis and other established species in this genus, further supporting novelty of this species. Noticeably, different sample treatment methods resulted in morphological variation of the apical pore complex (APC) of H. bohaiensis, which needs to be taken into account in future study.

Integration of the nuclease protection assay with sandwich hybridization (NPA-SH) for sensitive detection of Heterocapsa triquetra

Microalgae are photosynthetic microorganisms that function as primary producers in aquatic ecosystems. Some species of microalgae undergo rapid growth and cause harmful blooms in marine ecosystems. Heterocapsa triquetra is one of the most common bloom-forming species in estuarine and coastal waters worldwide. Although this species does not produce toxins, unlike some other Heterocapsa species, the high density of its blooms can cause significant ecological damage. We developed a H. triquetra species-specific nuclease protection assay sandwich hybridization (NPA-SH) probe that targets the large subunit of ribosomal RNA (LSU rRNA). We tested probe specificity and sensitivity with five other dinoflagellates that also cause red tides. Our assay detected H. triquetra at a concentration of 1.5×104 cells/mL, more sensitive than required for a red-tide guidance warning by the Korea Ministry of Oceans and Fisheries in 2015 (3.0×104 cells/mL). We also used the NPA-SH assay to monitor H. triquetra in the Tongyeong region of the southern sea area of Korea during 2014. This method could detect H. triquetra cells within 3 h. Our assay is useful for monitoring H. triquetra under field conditions.

Plate pattern clarification of the marine dinophyte Heterocapsa triquetra sensu Stein (Dinophyceae) collected at the Kiel Fjord (Germany)

One of the most common marine dinophytes is a species known as Heterocapsa triquetra. When Stein introduced the taxon Heterocapsa, he formally based the type species H.triquetra on the basionym Glenodinium triquetrum. The latter was described by Ehrenberg and is most likely a species of Kryptoperidinium. In addition to that currently unresolved nomenclatural situation, the thecal plate composition of H.triquetra sensu Stein (1883) was controversial in the past. To clarify the debate, we collected material and established the strain UTKG7 from the Baltic Sea off Kiel (Germany, the same locality as Stein had studied), which was investigated using light and electron microscopy, and whose systematic position was inferred using molecular phylogenetics. The small motile cells (18-26μm in length) had a biconical through fusiform shape and typically were characterized by a short asymmetrically shaped, horn-like protuberance at the antapex. A large spherical nucleus was located in the episome, whereas a single pyrenoid laid in the lower cingular plane. The predominant plate pattern was identified as apical pore complex (Po, cp?, X), 4', 2a, 6'', 6c, 5s, 5''', 2''''. The triradiate body scales were 254-306nm in diameter, had 6 ridges radiating from a central spine, 9 peripheral and 3 radiating spines, and 12 peripheral bars as well as a central depression in the basal plate. Our work provides a clarification of morphological characters and a new, validly published name for this important but yet formally undescribed species of Heterocapsa: H.steinii sp. nov.

Feeding by the newly described mixotrophic dinoflagellate Gymnodinium smaydae: Feeding mechanism, prey species, and effect of prey concentration

To investigate feeding by the newly described mixotrophic dinoflagellate Gymnodinium smaydae, we explored the feeding mechanism and the kinds of prey species that G. smaydae is able to feed on. In addition, we measured the growth and ingestion rates of G. smaydae on optimal and suboptimal algal prey Heterocapsa rotundata and Heterocapsa triquetra as a function of prey concentration. Among the 19 algal prey species offered, G. smaydae ingested only thecate dinoflagellates H. rotundata, H. triquetra, Heterocapsa sp., and Scrippsiella trochoidea. Among the peduncle-feeding dinoflagellates so far reported, G. smaydae is the only grazer that is able to feed on S. trochoidea and one of the two species that are able to feed on H. triquetra. However, G. smaydae did not feed on the raphidophyte Heterosigma akashiwo, the cryptophytes Teleaulax sp. and Rhodomonas salina, and the small dinoflagellate Amphidinium carterae which all the other peduncle-feeding dinoflagellates except Stoeckeria algicida are able to feed on. G. smaydae fed on algal prey using a peduncle after anchoring the prey by a tow filament. All Heterocapsa species supported high positive growth of G. smaydae, S. trochoidea only helped in merely maintaining the predator population. With increasing mean prey concentration, the growth and ingestion rates for G. smaydae on H. rotundata increased rapidly, but became saturated at concentrations of 455ngCml−1 (3500cellsml−1), while that on H. triquetra increased rapidly, but slowly at concentrations of 293ngCml−1 (945cellsml−1). The maximum specific growth rates (i.e., mixotrophic growth) of G. smaydae on H. rotundata and H. triquetra were 2.226d−1 and 1.053d−1, respectively, at 20°C under a 14:10h light–dark cycle of 20μEm−2s−1, while the growth rates (i.e., phototrophic growth) under the same light conditions without added prey were 0.005 to −0.051d−1. The maximum ingestion rates of G. smaydae on H. rotundata and H. triquetra were 1.59ngCgrazer−1d−1 (12.3cellsgrazer−1d−1) and 0.24ngCgrazer−1d−1 (0.8cellsgrazer−1d−1), respectively. The calculated grazing coefficients for G. smaydae on co-occurring H. rotundata or H. triquetra were up to 0.23h−1 or 0.02h−1, respectively (i.e., 21% or 2% of the population of H. rotundata or H. triquetra was removed by G. smaydae populations in 1h). The results of the present study suggest that G. smaydae can sometimes have a considerable grazing impact on the population of H. rotundata.

HETEROCAPSA ARCTICA SUBSP. FRIGIDA SUBSP. NOV. (PERIDINIALES, DINOPHYCEAE)-DESCRIPTION OF A NEW DINOFLAGELLATE AND ITS OCCURRENCE IN THE BALTIC SEA1

Characteristics important in identification of Heterocapsa species (i.e., thecal plate pattern, body scale structure, and shape and position of the nucleus and pyrenoid) are practically identical in the dinoflagellate investigated here and in Heterocapsa arctica T. Horig. described from the Canadian Arctic. Analysis of internal transcribed spacer (ITS) sequences confirms that the two dinoflagellates are very closely related; however, there is a clear difference in their size and shape. Our experiments show that the low-salinity Baltic Sea brackish water does not reduce the size of the marine H. arctica to match that of the Baltic Sea morphotype. On the basis of these dissimilarities in general morphology and its geographic isolation in the Baltic Sea, we consider our material sufficiently differentiated from the typical H. arctica to warrant the status of a new subspecies, H. arctica subsp. frigida subsp. nov. Being of a distinct cell shape, the occurrence of subsp. frigida has been recorded in Algaline phytoplankton monitoring data collected since 1993. Although it has never been responsible for high biomass blooms, it commonly occurs in spring in the Northern Baltic Proper and in the western Gulf of Finland, when the water temperatures are <5°C.

Expansion of potentially harmful algal taxa in a Georgia Estuary (USA)

Evidence is widespread that species of harmful algae are showing up in new locations and that toxic bloom events may be increasing in magnitude and frequency. These trends are sometimes but not exclusively associated with cultural eutrophication. On the southeast coast of the USA, harmful species, bloom events, and deleterious ecosystem impacts were restricted to eutrophic estuaries and adjacent shelf waters of North Carolina and Florida prior to 2000. Specifically, Georgia and South Carolina waters either lacked HAB species or contained unremarkable concentrations. Beginning in 2000, however, numerous HAB taxa were collected in South Carolina coastal ponds and estuaries, and were associated with fish kills there. The present study documents the new appearance of HAB species in Georgia, in the Skidaway estuary, which has been sampled weekly for 22 years. Four HAB taxa were initially present when sampling began in 1986–1987, five new species appeared sporadically over the next 15 years, and then seven additional new taxa appeared between 2002 and 2008. Eleven of the sixteen taxa were dinoflagellates (species of Cochlodinium, Dinophysis, Gyrodinium, Heterocapsa, Karenia, Karlodinium, Kryptoperidinium, and Prorocentrum), four were raphidophytes ( Chattonella, Fibrocapsa, and Heterosigma species), with one diatom ( Pseudo-nitzschia). Notably, only two species occurred at concentrations exceeding 10 3 cells ml −1: Heterosigma akashiwo and Heterocapsa rotundata. One species, H. akashiwo, was present every spring/summer and in sufficient concentrations to discern temporal trends. H. exhibited significant increases in annual mean and peak abundances over the 22-year sample period. This increasing trend was significantly correlated with nutrient concentrations, specifically NH 4 and DON. Whereas factors responsible for the initial appearance of new HAB taxa in these waters are unknown, the evidence suggests their establishment is related to cultural eutrophication. If current trends of increasing number and abundance of HAB species continue, estuaries in Georgia can expect to exhibit the detrimental ecological manifestations commonly observed elsewhere.

Cellular and body scale morphology ofHeterocapsa huensissp. nov. (Peridiniales, Dinophyceae) found in Hue, Vietnam

Cellular and body scale structure of a new armored dinoflagellate Heterocapsa huensis, collected from Hue, Vietnam were investigated. Morphology of motile cell was observed by light, fluorescent and scanning electron microscopy, and body scale structure was examined by whole mounts of transmission electron microscopy. Cells of H. huensis were ellipsoid with a spherical nucleus located in the posterior and multiple pyrenoids located above the nucleus; this arrangement was similar to that of Heterocapsa pygmaea. Transmission electron microscopy revealed ultrastructure of the body scales consisted of a rounded triangular basal plate and three-dimensional ornaments. Structure of the basal plate resembles that of Heterocapsa illdefina; however, the number of the peripheral spine is different from that of H. illdefina and this structure has never been reported from Heterocapsa species. A new Heterocapsa species, H. huensis Iwataki et Matsuoka sp. nov., is described based on positions of organelles and body scale ultrastructure.

Taxonomy and identification of the armored dinoflagellate genus Heterocapsa (Peridiniales, Dinophyceae)

The armored dinoflagellate genus Heterocapsa is composed of relatively small species, including a species responsible for harmful red tides, H. circularisquama. Some Heterocapsa species such as H. rotundata and H. triquetra have been well documented as red tide-forming species, but are not recognized as causing harmful effects. Following sequential shellfish mass mortalities in the coastal waters of western Japan due to H. circularisquama red tides, this species has attracted considerable interest. Many properties of this species, such as its distribution and growth charac- teristics, have been investigated to better understand the mechanisms involved in harmful red tide formation. Related to the need for unambiguous identification of H. circularisquama, several unidentified (or undescribed) Heterocapsa species, which must be distinguished from the harmful taxon, have been detected sympatrically in regions where H. cir- cularisquama blooms occur. However, the taxonomic affiliation of these Heterocapsa species have not yet been deter- mined because several characteristics of Heterocapsa (e.g. body scale ultrastructure) have not yet been reported from all described species due to the changing taxonomic criteria. After the taxonomic ambiguities in this genus were re- solved, cellular and body scale morphology of Heterocapsa were reinvestigated and several new species were described. In the present paper, the taxonomic history and morphological characters of the genus Heterocapsa are summarized.

High Incidence of Cyanobacterial Blooms along the Coast of the Cameroon Gulf of Guinea and their Effects on Human Health and Amenities

The coast of the Cameroon Gulf of Guinea (GoG) comprises areas like the, Idenau beach, Mudeka/Tiko creeks, Limbe estuary and Douala Lagoon most of which are in the immediate vicinity of industrial zones, logging stations and an oil refinery. They serve as centres for recreational sailing, fin and shellfish fisheries and sinks for the disposal of wastes. The coastline of the Cameroon GoG is generally characterised by low salinity due to high rainfall and a dense river network, which supplies freshwater. The peak salinity values recorded during the dry and rainy seasons were 20 and 12 respectively. High levels of organic nutrients ranging from 825.92 to 1671.26 ppm were recorded. Various species of Phormidium, Heterocapsa, Rivularia, Trichodesmium , and Lyngbya were identified. Water management in Cameroon is threatened with extensive and persistent noxious occurrences of harmful cyanobacterial blooms (HCBs) and they have had a significant impact on the utilization of aquatic resources. Blooms of Lyngbya have been incriminated in cases of severe dermatitis. Fish and shellfish consumers have also reported many acute cases of sudden abdominal cramps, headaches, vomits and diarrhoea. Recommendations are made and the need for systematic long-term research in collaboration with developed country scientists is elucidated. Keywords : GoG, HABs, HCBs, dry and rainy seasons, cyanobacteria, harmful, blooms > Tropical Freshwater Biology Vol. 15 2006: pp. 33-42

Heterocapsa psammophila sp. nov. (Peridiniales, Dinophyceae), a new sand‐dwelling marine dinoflagellate

SUMMARYA new armored dinoflagellate species, Heterocapsa psammophila Tamura, Iwataki et Horiguchi sp. nov. is described from Kenmin‐no‐hama beach, Hiroshima, Japan using light and electron microscopy. This dinoflagellate possesses the typical thecal plate arrangement of the genus Heterocapsa, Po, cp, 5′, 3a, 7′′, 6c, 5s, 5′′′, 2′′′′; and the 3‐D body scales of Heterocapsa on the plasma membrane. The cell shape is ovoidal. The spherical nucleus and the pyrenoid are situated in the hypotheca and the epitheca, respectively. The ultrastructure of H. psammophila is typical of dinoflagellates and the pyrenoid is invaginated by cytoplasmic tubules. H. psammophila is distinguished from all other hitherto‐described Heterocapsa species by the cell shape, the relative position of the nucleus and pyrenoid and the structure of the body scale. The habitat and behavior of this new species in culture suggest that the organism is truly a sand‐dwelling species.

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.

Investigations of body scales in twelve Heterocapsa species (Peridiniales, Dinophyceae), including a new species H. pseudotriquetra sp. nov.

The body scales of 12 Heterocapsa species including a new species, H. pseudotriquetra Iwataki, Gert Hansen & Fukuyo, sp. nov. were investigated by transmission electron microscopy to clarify ultrastructural differences. All species had scales with a body-scale structure consisting of a triradiate basal plate and a three-dimensional construction composed of vertically standing uprights or spines and horizontal bars. This basic scale structure is considered to be a generic characteristic. Finer scale details such as the shape and presence of a central hole in the basal plate, the numbers of uprights, bars and spines are considered to be species-specific. Two types of scales were found within clonal cultures of H. arctica and H. circularisquama and it is suggested that these represent ‘mature’ and ‘immature’ body scales. Intraspecific variation of the basal plate ‘reticulation’ was also observed. This variation was particularly evident in prolonged cultures of H. horiguchii and H. rotundata. The body-scale structure of 11 described species can be distinguished. However, two species, namely H. triquetra and H. pseudotriquetra, have the same scale morphology and are distinguished by differences in cell shape and LSU rDNA (25S rDNA) sequences.

Dinoflagellate community analysis of a fish kill using denaturing gradient gel electrophoresis

A molecular method using the polymerase chain reaction (PCR) amplification of small subunit gene sequences (18S rDNA) and denaturing gradient gel electrophoresis (DGGE) was used to determine both the population complexity and species identification of organisms in harmful algal blooms. Eighteen laboratory cultures of dinoflagellates, including Akashiwo, Gymnodinium, Heterocapsa, Karenia, Karlodinium, Pfiesteria, and Pfiesteria-like species were analyzed using dinoflagellate-specific oligonucleotide primers and DGGE. The method is sensitive and able to determine the number of species in a sample, as well as the taxonomic identity of each species, and is particularly useful in detecting differences between species of the same genus, as well as differences between morphologically similar species. Using this method, each of eight Pfiesteria-like species was verified as being clonal isolates of Pfiesteria piscicida. The sensitivity of dinoflagellate DGGE is approximately 1000 cells/ml, which is 100-fold less sensitive than real-time PCR. However, the advantage of DGGE lies in its ability to analyze dinoflagellate community structure without needing to know what is there, while real-time PCR provides much higher sensitivity and detection levels, if probes exist for the species of interest, attributes that complement DGGE analysis. In a blinded test, dinoflagellate DGGE was used to analyze two environmental fish kill samples whose species composition had been previously determined by other analyses. DGGE correctly identified the dominant species in these samples as Karlodinium micrum and Heterocapsa rotundata, proving the efficacy of this method on environmental samples. Toxin analysis of a clonal isolate obtained from the fish kill samples confirmed the presence of KmTx2, corroborating the earlier genetic identification of toxic K. micrum in the fish kill water sample.