Phytoplankton diversity explained by connectivity across a mesoscale frontal system in the open ocean.

Phytoplankton community composition is important in establishing ecosystem structure and function. Intuitively, we recognize that water movements must be important for modifying spatial gradients and plankton diversity. However, identifying boundaries and exchange between habitats in the open ocean is not straightforward. Here, we use the abundance of nine phytoplankton species closely sampled in a mesoscale frontal system in the northeastern North Sea as a proxy for community composition and explore the relationship between phytoplankton biogeography and transport patterns. Subsurface community distributions could be related to modeled patterns in water movement. A methodology for analyzing pelagic diversity that includes a representation of plankton community composition and an Eulerian connectivity tracer was developed, and the relative importance of connectivity and geographical distance for phytoplankton species composition analyzed. The connectivity tracer identifies timescales and dispersal barriers in the open ocean. Connectivity was found to be superior in explaining pelagic plankton diversity and found to be a prerequisite for understanding the pelagic phytoplankton composition. This approach is a valuable tool for establishing the link between ocean transports, ecosystem structure and biodiversity and for informing the placement of marine protected areas.

Ultrastructure and phylogeny of Parvodinium cunningtonii comb. nov. (syn. Peridiniopsis cunningtonii) and description of P. cunningtonii var. inerme var. nov. (Peridiniopsidaceae, Dinophyceae).

Two strains of peridinioids were isolated from a flooded stream near Aveiro, central Portugal, and examined by light microscopy, scanning electron microscopy and serial-section transmission electron microscopy. The two strains showed the same tabulation and cell shape as Peridiniopsis cunningtonii. One of the strains had lightly reticulated plates and spines in most hypothecal plates, matching the features of typical P. cunningtonii. The other strain showed smooth plates and consistently lacked spines in the apiculate hypotheca. The strains were similar in fine structure and had a central pyrenoid with a starch sheath and perforated by cytoplasmic channels. Details of the flagellar apparatus matched those known from Parvodinium, as did the remarkably long microtubular strand leading to an extruded peduncle that was visible in serial sections. Phylogenetic analyses based on partial LSU rDNA and the concatenated ribosomal operon placed the strain with the smooth hypotheca in a clade with Parvodinium species. The two strains grouped as closely related sister taxa in the partial LSU rDNA phylogeny. A new combination is proposed, Parvodinium cunningtonii comb. nov. and a new variety, Parvodinium cunningtonii var. inerme var. nov., is described.

Fine-structural characterization and phylogeny of Sphaerodinium (Suessiales, Dinophyceae), with the description of an unusual type of freshwater dinoflagellate cyst.

Two strains of Sphaerodinium were established from two mountain areas in Portugal and examined by light microscopy, scanning and transmission electron microscopy, and sequence analyses of nuclear-encoded SSU, ITS1-5.8S-ITS2 and LSU rDNA. Both strains were identified as S. polonicum var. tatricum on the basis of comparison with the original taxonomic descriptions within the genus. The two strains were nearly identical in morphology and ultrastructure, except for the presence of pseudograna-like thylakoid stacks within more rounded chloroplast lobes in one of them. Sexual reproduction occurred in culture batches and resting cysts with single or grouped processes with wide bases and distal platforms with slightly recurved margins were seen to develop by sudden retraction of planozygote cytoplasm. Morphological, fine-structural and molecular characters were compared with previously available information from S. cracoviense, allowing for a more robust characterization of the genus. Important characters include a type F eyespot, a pusule canal linking the transverse flagellar canal to a collecting chamber connected to regular pusular tubes, a ventral fibre extending from the proximal-right side of the longitudinal basal body, and a membranous, lamellar body with a honeycomb pattern near the flagellar base area. The latter two features are shared with Baldinia anauniensis.

The impact of urea on toxic diatoms - Potential effects of fertilizer silo breakdown on a Pseudo-nitzschia bloom.

In spring 2016, two silos containing liquid nitrogen-containing fertilizer collapsed on a harbor in Fredericia, Denmark. More than 2,750 tons of fertilizer spilled into inner Danish waters. A bloom of Pseudo-nitzschia occurred approximately one month after the incident. The bloom caused a 5-week quarantine of numerous mussel-harvesting areas along the eastern coast of Jutland. The levels of domoic acid measured up to 49 mg kg-1 in mussel meat after the bloom. In the months following the event, the species diversity of phytoplankton was low, while the abundance was high comprising few dominant species including Pseudo-nitzschia. The main part of the liquid nitrogen-containing compound was urea, chemically produced for agricultural use. To investigate the potential impact of urea on Pseudo-nitzschia, four strains, including one strain of P. delicatissima, two of P. seriata and one of P. obtusa, were exposed each to three concentrations of urea in a batch culture experiment: 10 µM, 20 µM and 100 µM N urea, and for comparison one concentration of nitrate (10 µM). Nitrate, ammonium, and urea were metabolized at different rates. Pseudo-nitzschia obtusa produced domoic acid and grew best at low urea concentrations. Both P. seriata strains had a positive correlation between urea concentration and growth rate, and the highest growth rate in the nitrate treatment. One strain of P. seriata produced domoic acid peaking at low N loads (10 µM N urea and 10 µM N nitrate). In conclusion, the ability to adapt to the available nitrogen source and retain a high growth rate was exceedingly varying and not only species-specific but also strain specific.

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.

Studies on Woloszynskioid Dinoflagellates X: Ultrastructure, Phylogeny and Colour Variation in Tovellia rubescens n. sp. (Dinophyceae).

The external morphology and internal cell fine structure of a new species of Tovelliaceae, Tovellia rubescens n. sp., is described. Phylogenetic analyses based on partial LSU rDNA sequences place the new species in a clade containing Tovellia species that accumulate red pigments and identify T. aveirensis as its closest known relative. Cells of T. rubescens n. sp. were mostly round and had the cingulum located near the middle, with its ends displaced about one cingular width. Small numbers of distinctly flat cells appeared in culture batches; their significance could not be determined. Cells of the new species in culture batches progressively changed from a yellowish-green, mainly due to chloroplast colour, to a reddish-brown colour that appeared associated with lipid bodies. The switch to a reddish colour happened earlier in batches grown in medium lacking sources of N or P. Pigment analyses by HPLC-MS/MS revealed the presence of astaxanthin and astaxanthin-related metabolites in the new species, but also in T. aveirensis, in which a reddish colour was never observed. The chloroplast arrangement of T. rubescens n. sp. resembled that of T. aveirensis, with lobes radiating from a central pyrenoid complex. The flagellar apparatus and pusular system fell within the general features described from other Tovelliaceae. A row of microtubules interpretable as a microtubular strand of the peduncle was present. Spiny resting cysts with red contents and an ITS sequence identical to that of cultured material of the new species were found in the original locality.

A-, B- and C-type prymnesins are clade specific compounds and chemotaxonomic markers in Prymnesium parvum.

Harmful blooms formed by planktonic microalgae (HABs) in both freshwater and coastal waters regularly lead to severe mortalities of fish and invertebrates causing substantial economic losses of marine products worldwide. The mixotrophic haptophyte Prymnesium parvum is one of the most important microalgae associated with fish kills. Here 26 strains of P. parvum with a wide geographical distribution were screened for the production of prymnesins, the suspected causative allelochemical toxins. All investigated strains produced prymnesins, indicating that the toxins play an important role for the organism. The prymnesins can be classified into three types based on the length of the carbon backbone of the compound and each algal strain produced only one of these types. Biogeographical mapping of the prymnesin distribution indicated a global distribution of each type. In addition, phylogenetic analyses based on internal transcribed spacer (ITS) sequences revealed monophyletic origin of all prymnesin types and clades could therefore be defined based on the toxic compound. It might be that evolution of new species within the P. parvum species complex is driven by changes in toxin type or that they are a result of it. Such a correlation between chemotype and phylotype has never been documented before for a harmful microalga. Chemotaxonomy and ITS-type classification may thus be used to further delimit the P. parvum species complex.

Baffinella frigidus gen. et sp. nov. (Baffinellaceae fam. nov., Cryptophyceae) from Baffin Bay: Morphology, pigment profile, phylogeny, and growth rate response to three abiotic factors.

Twenty years ago an Arctic cryptophyte was isolated from Baffin Bay and given strain number CCMP2045. Here, it was described using morphology, water- and non-water soluble pigments and nuclear-encoded SSU rDNA. The influence of temperature, salinity, and light intensity on growth rates was also examined. Microscopy revealed typical cryptophyte features but the chloroplast color was either green or red depending on the light intensity provided. Phycoerythrin (Cr-PE 566) was only produced when cells were grown under low-light conditions (5 µmol photons · m-2  · s-1 ). Non-water-soluble pigments included chlorophyll a, c2 and five major carotenoids. Cells measured 8.2 × 5.1 µm and a tail-like appendage gave them a comma-shape. The nucleus was located posteriorly and a horseshoe-shaped chloroplast contained a single pyrenoid. Ejectosomes of two sizes and a nucleomorph anterior to the pyrenoid were discerned in TEM. SEM revealed a slightly elevated vestibular plate in the vestibulum. The inner periplast component consisted of slightly overlapping hexagonal plates arranged in 16-20 oblique rows. Antapical plates were smaller and their shape less profound. Temperature and salinity studies revealed CCMP2045 as stenothermal and euryhaline and growth was saturated between 5 and 20 µmol photons · m-2  · s-1 . The phylogeny based on SSU rDNA showed that CCMP2045 formed a distinct clade with CCMP2293 and Falcomonas sp. isolated from Spain. Combining pheno- and genotypic data, the Arctic cryptophyte could not be placed in an existing family and genus and therefore Baffinellaceae fam. nov. and Baffinella frigidus gen. et sp. nov. were proposed.

The Rainwater Rock-pool Dinoflagellate Nottbeckia ochracea gen. et comb. nov. (syn.: Hemidinium ochraceum) - A Fine-structural and Molecular Study with Emphasis on the Motile Stage.

The rarely recorded dinoflagellate Hemidinium ochraceum Levander was isolated from a rainwater rock-pool in Finland and the motile stage studied by light- and electron microscopy and molecular sequencing. The ultrastructure of the motile stage revealed several features not previously observed in other dinoflagellates, e.g. thecal plates with inwards 'knobs' and a peculiar corrugated left sulcal plate. The apparent lack of the R2 and R4 flagellar roots was also unusual. The typical plate tabulation was 4', 2a, 8″, cx, 4c,?s, 3‴, cy, 1p, 2″″, cx and cy being extra plates associated with the cingulum, but intraspecific variations were observed in shape and number of plates. Phylogenetic analyses of nuclear-encoded LSU rDNA and concatenation of LSU and SSU rDNA sequences showed that H. ochraceum was only distantly related to Hemidinium nasutum (the type species of Hemidinium). The immotile coccoid stage resembled Gloeodinium montanum and a weakly supported relationship between the two was only inferred in the analyses based on LSU rDNA. Hemidinium ochraceum showed some resemblance to Rufusiella insignis sensu Richards, but the true identity of this species and of genus Rufusiella is uncertain. A new genus, Nottbeckia, is erected to accommodate H. ochraceum, as Nottbeckia ochracea gen. et comb. nov.

Ultrastructure and Phylogeny of Kirithra asteri gen. et sp. nov. (Ceratoperidiniaceae, Dinophyceae) - a Free-Living, Thin-Walled Marine Photosynthetic Dinoflagellate from Argentina.

A gymnodinioid photosynthetic dinoflagellate was isolated from Argentina and examined by light and electron microscopy and analysis of nuclear-encoded LSU rDNA. Kirithra asteri gen. et sp. nov. was proposed as morphology and molecular phylogeny separated this dinoflagellate from others within the family Ceratoperidiniaceae. Cells were surrounded by a hyaline amphiesma comprising polygonal vesicles. Each vesicle contained a honeycomb and a trilaminar structure. An anterior sulcal extension ending in a complete circle formed the apical structure complex (ASC), which characterizes Ceratoperidiniaceae. The ASC comprised three rows of vesicles. The nucleus was located in the hypocone, and several large, irregularly shaped vesicles were present in the epi- and hypocone. Chloroplasts were surrounded by three membranes, and grana-like arrangements of thylakoids were observed in one strain used for ultrastructural study. The cell centre contained 1-3 multiple-stalked pyrenoids and membrane-bound vesicles containing tile-like structures surrounded each pyrenoid. Two pusules with collecting chambers and associated vesicles branched off each of the flagellar canals. The flagellar apparatus featured a ventral connective between the amphiesma and the R1 root, and almost opposite basal bodies, rarely seen in dinoflagellates. This was the first ultrastructural study of a species within Ceratoperidiniaceae.

Effects of irradiance and prey deprivation on growth, cell carbon and photosynthetic activity of the freshwater kleptoplastidic dinoflagellate Nusuttodinium (= Gymnodinium) aeruginosum (Dinophyceae).

The freshwater dinoflagellate Nusuttodinium aeruginosum lacks permanent chloroplasts. Rather it sequesters chloroplasts as well as other cell organelles, like mitochondria and nuclei, from ingested cryptophyte prey. In the present study, growth rates, cell production and photosynthesis were measured at seven irradiances, ranging from 10 to 140 µmol photons m-2s-1, when fed the cryptophyte Chroomonas sp. Growth rates were positively influenced by irradiance and increased from 0.025 d-1 at 10 µmol photons m-2s-1 to maximum growth rates of ~0.3 d-1 at irradiances ≥ 40 µmol photons m-2s-1. Similarly, photosynthesis ranged from 1.84 to 36.9 pg C cell-1 h-1 at 10 and 140 µmol photons m-2s-1, respectively. The highest rates of photosynthesis in N. aeruginosum only corresponded to ~25% of its own cell carbon content and estimated biomass production. The measured rates of photosynthesis could not explain the observed growth rates at high irradiances. Cultures of N. aeruginosum subjected to prey starvation were able to survive for at least 27 days in the light. The sequestered chloroplasts maintained their photosynthetic activity during the entire period of starvation, during which the population underwent 4 cell divisions. This indicates that N. aeruginosum has some control of the chloroplasts, which may be able to replicate. In conclusion, N. aeruginosum seems to be in an early stage of chloroplast acquisition with some control of its ingested chloroplasts.

Evolutionary transition towards permanent chloroplasts? - Division of kleptochloroplasts in starved cells of two species of Dinophysis (Dinophyceae).

Species within the marine toxic dinoflagellate genus Dinophysis are phagotrophic organisms that exploit chloroplasts (kleptochloroplasts) from other protists to perform photosynthesis. Dinophysis spp. acquire the kleptochloroplasts from the ciliate Mesodinium rubrum, which in turn acquires the chloroplasts from a unique clade of cryptophytes. Dinophysis spp. digest the prey nuclei and all other cell organelles upon ingestion (except the kleptochloroplasts) and they are therefore believed to constantly acquire new chloroplasts as the populations grow. Previous studies have, however, indicated that Dinophysis can keep the kleptochloroplasts active during long term starvation and are able to produce photosynthetic pigments when exposed to prey starvation. This indicates a considerable control over the kleptochloroplasts and the ability of Dinophysis to replicate its kleptochloroplasts was therefore re-investigated in detail in this study. The kleptochloroplasts of Dinophysis acuta and Dinophysis acuminata were analyzed using confocal microscopy and 3D bioimaging software during long term starvation experiments. The cell concentrations were monitored to confirm cell divisions and samples were withdrawn each time a doubling had occurred. The results show direct evidence of kleptochloroplastidic division and that the decreases in total kleptochloroplast volume, number of kleptochloroplasts and number of kleptochloroplast centers were not caused by dilution due to cell divisions. This is the first report of division of kleptochloroplasts in any protist without the associated prey nuclei. This indicates that Dinophysis spp. may be in a transitional phase towards possessing permanent chloroplasts, which thereby potentially makes it a key organism to understand the evolution of phototrophic protists.

Dynamics of Sequestered Cryptophyte Nuclei in Mesodinium rubrum during Starvation and Refeeding.

The marine mixotrophic ciliate Mesodinium rubrum is known to acquire chloroplasts, mitochondria, nucleomorphs, and nucleus from its cryptophyte prey, particularly from species in the genera, Geminigera and Teleaulax. The sequestered prey nucleus and chloroplasts are considered to support photosynthesis of M. rubrum. In addition, recent studies have shown enlargement of the retained prey nucleus in starved M. rubrum and have inferred that enlargement results from the fusion of ingested prey nuclei. Thus far, however, little is known about the mechanism underlying the enlargement of the prey nucleus in M. rubrum. Here, we conducted starvation and refeeding studies to monitor the fate of prey nuclei acquired by M. rubrum when feeding on Teleaulax amphioxeia and to explore the influence of the retained prey nucleus on photosynthesis of M. rubrum. Results indicate that enlargement of the prey nucleus does not result from fusion of nuclei. Furthermore, the enlarged prey nucleus does not appear to divide during cell division of M. rubrum. The presence of a prey nucleus significantly affected photosynthetic performance of M. rubrum, while the number of retained chloroplasts had little influence on rate of carbon fixation. We interpret results within the context of a model that considers the dynamics of ingested prey nuclei during division of M. rubrum.

Interspecific Competition Study Between Pseudochattonella farcimen and P. verruculosa (Dictyochophyceae)-Two Ichthyotoxic Species that Co-occur in Scandinavian Waters.

The genus Pseudochattonella has become a frequent component of late winter-early spring phytoplankton community in Scandinavian waters, causing extensive fish kills and substantial economic losses. One of currently two recognised species, P. farcimen, is often abundant prior to the diatom spring bloom. Recent field studies have revealed that P. farcimen and P. verruculosa have a period of overlap in their temperature ranges and thus their seasonal occurrences. Using laboratory cultures, we investigated the seasonal succession and growth of P. farcimen and P. verruculosa in both mono- and mixed-culture using the recently developed Pseudochattonella 'qPCR subtraction method', which for the first time allowed the simultaneous enumeration of these morphologically indistinguishable species in mixed assemblages. We examined how these species interacted over four different temperatures (5, 8, 11 and 15 °C). The observed growth rates and cell yields varied with temperature revealing their preferred temperature optima. P. farcimen was able to achieve positive net growth over all temperatures, while P. verruculosa failed to grow below 11 °C. Growth responses were statistically different between mono- and mixed-cultures with the outcome of these interactions being temperature-dependent. Nutrients (nitrate and phosphate) and pH levels were also measured throughout the growth experiments to better understand how these factors influenced growth of both species. P. verruculosa was shown to be less sensitive to high pH as growth ceased at pH 9.1, whereas P. farcimen stopped growing at pH 8.4. Understanding the influence of abiotic factors (e.g. temperature, pH and competition) on growth rates allows for a better understanding and prediction of phytoplankton community dynamics.

A quantitative real-time PCR assay for identification and enumeration of the occasionally co-occurring ichthyotoxic Pseudochattonella farcimen and P. verruculosa (Dictyochophyceae) and analysis of variation in gene copy numbers during the growth phase of single and mixed cultures.

The ichthyotoxic genus Pseudochattonella forms recurrent extensive blooms in coastal waters in Japan, New Zealand and Northern Europe. It comprises of two morphologically similar species, P. verruculosa and P. farcimen, which complicates visual species identification and enumeration of live and fixed material. Primers designed previously could not quantitatively distinguish species in mixed assemblages. To address this issue we developed two primer sets: one revealed itself to be genus specific for Pseudochattonella and the other species-specific for P. verruculosa. By subtracting cell estimates for P. verruculosa from combined results we could calculate cell numbers for P. farcimen. This approach has overcome the challenges posed by the very limited sequence availability and low gene variability between the two species. The qPCR assay was extensively tested for specificity, efficiency and sensitivity over an entire growth cycle in both single and mixed assemblages. Comparison of cell abundance estimates obtained by qPCR assay and microscopy showed no statistically significant difference until stationary and death phases. The assay was also tested on environmental samples collected during a small Pseudochattonella bloom in Denmark in March-April 2015. It was impossible to distinguish P. farcimen and P. verruculosa by light microscopy but qPCR showed both species were present. The two methods provided nearly identical cell numbers but the assay provided discrimination and enumeration of both species.

The ichthyotoxic genus Pseudochattonella (Dictyochophyceae): Distribution, toxicity, enumeration, ecological impact, succession and life history - A review.

The marine genus Pseudochattonella is a recent addition to the list of fish killing microalgae. Currently two species are recognised (viz. P. verruculosa and P. farcimen) which both form recurrent coastal blooms sometimes overlapping in space and time. These events and their ecological and economic consequences have resulted in great interest and concern from marine biologists and the aquaculture industry. Since the first recorded blooms in Japanese (late 1980s), Scandinavian (1993) and Chilean (2004) waters numerous studies have focused on understanding the causative means of the fish killing. Mortality is probably due to Pseudochattonella discharging mucocysts that cause gill irritation and damage to the fish fills. Here, a review is provided of the literature on Pseudochattonella that covers the last ca. 25 years and focus on a number of topics relevant to understanding the general biology of the genus including ways to distinguish the two species. The literature addressing biogeography and known harmful events is evaluated and based on these findings an updated distribution map is proposed. P. farcimen is presently restricted to North European waters. Despite being very difficult to delineate based on morphology alone the two Pseudochattonella species seem to have separate growth optima. In laboratory experiments P. verruculosa consistently has higher temperature growth optima compared to P. farcimen though periods of overlap have been noted in the field. The review ends by proposing five areas with knowledge gaps and each of these could form the basis of future studies.

Fine-structural characterization and phylogeny of Peridinium polonicum, type species of the recently described genus Naiadinium (Dinophyceae).

Peridinium polonicum is a freshwater peridinioid with an unusual tabulation that includes one or two anterior intercalary plates in the mid-dorsal axis, and in such a low position that it seems inset in precingular Plate 4. Although the species has been classified in both Peridinium and Peridiniopsis, evidence from nucleotide sequences consistently shows that its closest relatives are within the Scrippsiella group. The genus Naiadinium Carty has been recently described with P. polonicum as its type species. However, Naiadinium was separated from other peridinioids only on the basis of shape and plate arrangements and these characters do not allow reliable determination of its closest phylogenetic relatives. Serial section fine-structural analysis revealed the presence of a small peduncle supported by a conspicuous microtubular basket that extended far into the cell; a complex pusular system that included a collecting chamber from which about 70 pusular tubes radiated; a flagellar apparatus with general peridinioid characters but with an unusually large distance of nearly 700 nm between basal bodies. An ITS1-5.8S-ITS2 rDNA-based phylogenetic analysis grouped, with high statistical support, Naiadinium polonicum with three species currently placed in Scrippsiella, viz. S. irregularis, S. precaria and S. ramonii.

Development of a multiplex real-time qPCR assay for simultaneous enumeration of up to four marine toxic bloom-forming microalgal species.

Harmful algal blooms (HAB) pose serious economic and health risks worldwide. Current methods of identification require high levels of taxonomic skill and can be highly time-consuming thus limiting sample throughput. So, new rapid and reliable methods for detection and enumeration of HAB species are required. Here we describe a high-throughput, multiplex-qPCR (M-qPCR) method using hydrolysis probe technology for the simultaneous detection of four HAB species commonly found in many coastal areas worldwide: Alexandrium tamarense, Karenia mikimotoi, Karlodinium veneficum and Prymnesium parvum. Primers and probes were species-specific and highly efficient when tested in simplex. Species were then added in succession and the assay conditions adjusted until all four species could be quantitatively evaluated simultaneously. Enumeration accuracy of the M-qPCR assay as a monitoring tool was evaluated using spiked natural environmental samples from Danish coastal waters. Comparison of estimates of cell abundances obtained by the M-qPCR technique with those obtained by light microscopy (Sedgwick Rafter technique) showed no statistically significant difference across a range of concentrations. We were also able to identify and enumerate target cells that would be below the detection limit of light microscopy making this a suitable method for early bloom detection or for low biomass species. With the development of molecular probes for a greater number of algal species M-qPCR will be of great benefit to phytoplankton monitoring programmes and the aquaculture industry worldwide.

Choice of pore size can introduce artefacts when filtering picoeukaryotes for molecular biodiversity studies.

Published results of studies based on samples size fractionated by sequential filtration (e.g. 0.2-3 µm) indicate that many ciliate, dinoflagellate and rhizarian phylotypes are found among marine picoeukaryotes. This is somewhat surprising as these protists are typically known as being large organisms (often >10 µm) and no picoplanktonic species have so far been identified. Here, the abundances of ciliate and dinoflagellate phylotypes in published molecular studies of picoeukaryotes are shown to correlate negatively with the pore size chosen for the end filter in the sequential filtrations (i.e. the filter used to collect the microbial biomass). This suggests that extracellular DNA adhering to small particles may be the source of ciliate and dinoflagellate phylotypes in picoplanktonic size fractions. This hypothesis was confirmed using real-time qPCR, which revealed significantly less dinoflagellate 18S rDNA in a 0.8-3-µm size fraction compared to 0.2-3 µm. On average, the abundance of putative extracellular phylotypes decreased by 84-89 % when a 0.8- µm end filter was used rather than a 0.2-µm end filter. A 0.8-µm filter is, however, not sufficient to retain all picoeukaryotic cells. Thus, selection of filter pore size involves a trade-off between avoiding artefacts generated by extracellular DNA and sampling the entire picoeukaryotic community. In contrast to ciliate and dinoflagellate phylotypes, rhizarian phylotypes in the picoplankton size range do not display a pattern consistent with an extracellular origin. This is likely due to the documented existence of picoplanktonic swarmer cells within this group.

Using nuclear-encoded LSU and SSU rDNA sequences to identify the eukaryotic endosymbiont in Amphisolenia bidentata (Dinophyceae).

The marine dinoflagellate Amphisolenia bidentata possesses complete intracellular symbionts of prokaryotic and eukaryotic origin. This was confirmed ultrastructurally little over 20 years ago when it was showed that the eukaryotic endosymbiont had a nucleus, a chloroplast and mitochondria. We collected Amphisolenia bidentata cells in the Indian Ocean and the identity of the eukaryotic endosymbionts was investigated using both microscopical and molecular methods. Individual specimens of Amphisolenia bidentata were identified by light microscopy and selected for single-cell PCR. Host and endosymbiont nuclear-encoded LSU and SSU rDNA sequences were determined by PCR cloning. Blast searches showed the endosymbiont LSU sequence to have affinity to Pelagophyceae, an algal class within Chromalveolata that also includes dinoflagellates. Since more SSU rDNA sequences from pelagophytes are available we performed a SSU based phylogeny of chromalveolates. The eukaryotic endosymbiont clustered within a clade comprising flagellated and coccoid pelagophytes whereas Amphisolenia bidentata formed a sister taxon to other dinophysioids. Molecular data therefore resolved the endosymbiont in A. bidentata being a pelagophyte and thus identified the ninth novel chloroplast type in dinoflagellates and a new species association. Based on sequence divergence estimates and phylogenetic inference the endosymbiont in A. bidentata likely represents an undescribed genus of pelagophytes.