Picoeukaryotic plankton diversity at the Helgoland time series site as assessed by three molecular methods.

We analyzed picoeukaryote assemblages in the German Bight at the Helgoland time series site by sequencing cloned eukaryotic 18S rRNA genes in six genetic libraries plus one library from the Orkney Islands from a cruise of opportunity. The libraries were constructed from environmental samples collected at different periods of the year. The same samples were also analyzed using a fingerprinting technique, single-strand conformational polymorphism (SSCP), and DNA microarrays with class-level oligonucleotide probes. One hundred unique clones were analyzed from each library, thus insuring over 85% coverage of the library. The V4 region of the 18S rRNA gene was sequenced from each of these clones, thus providing the most discrimination among the clones. The nonphotosynthetic picoeukaryotic component dominated over the photosynthetic one and was represented by the ciliates at 45% and group II alveolates at 42%. Prasinophytes dominated the photosynthetic group at 40%, but other picoplankton groups, such as bolidomonads and chrysophytes, were also present. Totally novel groups were found in the cryptomonads and in the dinoflagellates. A new algal group sister to the cryptophyte nuclear gene and the glaucocystophytes was also found. These three groups have been found in other picoeukaryotic planktonic clone libraries. SSCP analyses at closer time intervals suggest that clone libraries should be made at weekly intervals if succession in the picoeukaryotic plankton community is to be monitored accurately. A comparison of annual samples suggests thatthere appears to be an annual cycle with regard to species composition. Microarray analysis supported the clone library data and offered a faster means of community analysis, which can be performed with similar accuracy and with higher throughput for a more in-depth analysis.

Improved erythrocyte lysis assay in microtitre plates for sensitive detection and efficient measurement of haemolytic compounds from ichthyotoxic algae.

Haemolytic substances produced by ichthyotoxic algae often are unknown in molecular structure or specific mechanism of toxicity. Detection and quantification of such substances are dependent on bioassays, using markers that are sensitive for haemolytic impairment and generation of a recordable response. The erythrocyte lysis assay (ELA) represents an advantageous bioassay in this respect, because the lytic response can be measured photometrically by the amount of released haemoglobin. The aim of the present study was to establish an improved assay based on the ELA principle, for sensitive determination of haemolytic substances of microalgae and for high sample throughput. For this purpose we adapted the ELA to a 96-well microtitre plate format, which significantly reduced the sample volumes and allowed rapid processing of samples. Further improvement was achieved by measuring absorption of lysed erythrocytes at 414 nm, which significantly increased the sensitivity of the ELA compared to the measurements at 540 nm that are usually applied in this type of assay. Using carp (Cyprinus carpio) erythrocytes it was possible to detect haemolysis induced by 4 microg ml(-1) of saponin and as little as two haemolytic Alexandrium tamarense cells. It is suggested that this improved ELA in microtitre plates be used as a low-cost monitoring tool for detection and analysis of potential harmful algae. Furthermore, this ELA can be useful as a sensitive screening system for substances of pharmacological interest, e.g. selectively acting cytolytic antibiotics.

A simple and highly efficient fixation method for Chrysochromulina polylepis (Prymnesiophytes) for analytical flow cytometry.

BACKGROUND: To study the fragile Prymnesiophyte species Chrysochromulina polylepis by flow cytometry (FC), we needed an effective fixation method. This method must guarantee a high yield of fixed cells to achieve acceptable measurement times by FC and to allow quick processing of many samples. Moreover, we wanted a method that allows for storage of fixed samples when FC analysis cannot be done immediately. METHODS: Different aldehydes and methanol were tested at different final concentrations. Gravity sedimentation and centrifugation were applied to achieve higher cell concentrations. Storage of fixed samples was tested under different conditions. RESULTS: 0.25% glutaraldehyde (GA) fixation yielded a recovery rate of about 90%. The signals obtained by FC analysis were excellent. It is possible to centrifuge GA-fixed cells and to store them for several weeks. CONCLUSIONS: GA is the fixative of choice for FC analysis of C. polylepis (and possibly other small delicate species) because it yielded highly significant recovery rates and high-quality FC signals. Cells can be centrifuged to increase the cell concentration, thereby achieving short measurement times with FC. The possibility of long-term storage of fixed cells presents an additional advantage if FC analysis cannot be done immediately.

Oligonucleotide probes for the identification of three algal groups by dot blot and fluorescent whole-cell hybridization.

Photosynthetic pico- and nanoplankton dominate phytoplankton biomass and primary production in the oligotrophic open ocean. Species composition, community structure, and dynamics of the eukaryotic components of these size classes are poorly known primarily because of the difficulties associated with their preservation and identification. Molecular techniques utilizing 18S rRNA sequences offer a number of new and rapid means of identifying the picoplankton. From the available 18S rRNA sequence data for the algae, we designed new group-specific oligonucleotide probes for the division Chlorophyta, the division Haptophyta, and the class Pelagophyceae (division Heterokonta). Dot blot hybridization with polymerase chain reaction amplified target rDNA and whole-cell hybridization assays with fluorescence microscopy and flow cytometry were used to demonstrate probe specificity. Hybridization results with representatives from seven algal classes supported the phylogenetic affinities of the cells. Such group- or taxon-specific probes will be useful in examining community structure, for identifying new algal isolates, and for in situ detection of these three groups, which are thought to be the dominant algal taxa in the oligotrophic regions of the ocean.

Phylogenetic position of Cryothecomonas inferred from nuclear-encoded small subunit ribosomal RNA.

The systematic position of the genus Cryothecomonas has been determined from an analysis of the nuclear-encoded small subunit ribosomal RNA gene of Cryothecomonas longipes and two strains of Cryothecomonas aestivalis. Our phylogenetic trees inferred from maximum likelihood, distance and maximum parsimony methods robustly show that the genus Cryothecomonas clusters within the phylum Cercozoa, and is related to the sarcomonad flagellate Heteromita globosa. Morphological data supporting the taxonomic placement of Cryothecomonas near the sarcomonad flagellates has been compiled from the literature. The high number of nucleotide substitutions found between two morphologically indistinguishable strains of Cryothecomonas aestivalis suggests the possibility of cryptic species within Cryothecomonas aestivalis.

16S rRNA Targeted Probes for the Identification of Bacterial Strains Isolated from Cultures of the Toxic Dinoflagellate Alexandrium tamarense.

A BSTRACTBacteria have been implicated in the production of paralytic shellfish poison (PSP) toxins, which are normally associated with bloom-forming algal species, specifically toxic dinoflagellate algae. To clarify the role that these bacteria may play in the production of PSP toxins, it is desirable to identify and localize the bacteria associated with the dinoflagellates. 16S rRNA-targeted probes offer the possibility for both, and thus, probes have been made to putatively toxigenic bacteria isolated from the PSP-related dinoflagellate Alexandrium tamarense and tested for their specificity in dot blot and in situ hybridization experiments.

Ribosomal RNA analysis indicates a benthic pennate diatom ancestry for the endosymbionts of the dinoflagellates Peridinium foliaceum and Peridinium balticum (Pyrrhophyta).

The establishment of chloroplasts as cellular organelles in the dinoflagellate, heterokont (stramenopile), haptophyte, and cryptophyte algae is widely accepted to have been the result of secondary endosymbiotic events, that is, the uptake of a photosynthetic eukaryote by a phagotrophic eukaryote. However, the circumstances that promote such associations between two phylogenetically distinct organisms and result in the integration of their genomes to form a single functional photosynthetic cell is unclear. The dinoflagellates Peridinium foliaceum and Peridinium balticum are unusual in that each contains a membrane-bound eukaryotic heterokont endosymbiont. These symbioses have been interpreted, through data derived from ultrastructural and biochemical investigations, to represent an intermediate stage of secondary endosymbiotic chloroplast acquisition. In this study we have examined the phylogenetic origin of the P. foliaceum and P. balticum heterokont endosymbionts through analysis of their nuclear small subunit ribosomal RNA genes. Our analyses clearly demonstrate both endosymbionts are pennate diatoms belonging to the family Bacillariaceae. Since members of the Bacillariaceae are usually benthic, living on shallow marine sediments, the manner in which establishment of a symbiosis between a planktonic flagellated dinoflagellate and a bottom-dwelling diatom is discussed. In particular, specific environmentally-associated life strategy stages of the host and symbiont, coupled with diatom food preferences by the dinoflagellate, may have been vital to the formation of this association.

Evolution of the diatoms (Bacillariophyta). IV. A reconstruction of their age from small subunit rRNA coding regions and the fossil record.

Small subunit ribosomal RNA (ssu rRNA) coding regions from 30 diatoms, 3 oomycetes, and 6 pelagophytes were used to construct linearized trees, maximum-likelihood trees, and neighbor-joining trees inferred from both unweighted and weighted distances. Stochastic accumulation of sequence substitutions among the diatoms was assessed with relative rate tests. Pennate diatoms evolved relatively slowly but within the limits set by a stochastic model; centric diatoms exceeded those limits. A rate distribution test was devised to identify those taxa showing an aberrant distribution of base substitutions within the ssu rRNA coding region. First appearance dates of diatom taxa from the fossil record were regressed against their corresponding branch lengths to infer the average and earliest possible age for the origin of the diatoms, the pennate diatoms, and the centric diatom order Thalassiosirales. Our most lenient age estimate (based on the median-evolving diatom taxon in the maximum-likelihood tree or on the average branch length in a linearized tree) suggests that their average age is approximately 164-166 Ma, which is close to their earliest fossil record. Both calculations suggest that it is unlikely that diatoms existed prior to 238-266 Ma. Rate variation among the diatoms' ssu rRNA coding regions and uncertainties associated with the origin of extant taxa in the fossil record contribute significantly to the variation in age estimates obtained. Different evolutionary models and the exclusion of fast or slow evolving taxa did not significantly affect age estimates; however, the inclusion of aberrantly fast evolving taxa did. Our molecular clock calibrations indicate that the rRNA coding regions in the diatoms are evolving at approximately 1% per 18 to 26 Ma, which is the fastest substitution rate reported in any pro- or eukaryotic group of organisms to date.

Evolution of the diatoms (Bacillariophyta). II. Nuclear-encoded small-subunit rRNA sequence comparisons confirm a paraphyletic origin for the centric diatoms.

A phylogeny of the diatoms was inferred from comparisons of nuclear-encoded small subunit ribosomal RNA coding regions using maximum likelihood, weighted maximum parsimony, and neighbor-joining distance methods with Jukes and Cantor, Kimura, Gamma, van de Peer, and LogDet evolutionary models. Analyses of 30 taxa in 11 orders recovered two clades (Clades I and II). Neither of these clades correspond to the three classes of diatoms presently recognized or to the traditionally recognized radially symmetrical centric diatoms or bilaterally symmetrical pennate diatoms. All analyses show that the centric diatoms are a paraphyletic lineage. Tests of alternative phylogenies that address existing hypotheses regarding diatom systematics with the maximum likelihood and maximum parsimony methods support the two clades. Clade I is defined by centric diatom orders with specialized tubes, termed labiate processes, located peripherally in the cell wall. Clade II contains (1) bi(multi)polar centric diatoms with centrally located labiate processes, (2) centric diatoms with other central tubes termed strutted processes, and (3) pennate diatoms. Morphological evidence from fossil assemblages and cytological architecture support the results of the molecular analyses, whereas morphological features of extant diatoms are too derived to resolve the deeper branches in the tree.

Phylogenetic position of the Chromista plastids based on small subunit rRNA coding regions.

The kingdom Chromista contains eukaryotic organisms with tubular mastigonemes on the leading flagellum of their bi-flagellated stages, and plastids within a chloroplast endoplasmic reticulum (CER). The complex series of events leading to the formation of the CER is hypothesized to have occurred only once. Thus, all organisms with plastid-CER connections are believed to be monophyletic and derived from a single secondary endosymbiotic event. Analyses of sequence data from the 16s rRNA gene from three of the four Chromista pigmented classes indicate that these algae are not monophyletic. The validity of the kingdom Chromista and the number of secondary plastid endosymbioses are questioned.