New Insights into Plagiogrammaceae (Bacillariophyta) Based on Multigene Phylogenies and Morphological Characteristics with the Description of a New Genus and Three New Species

Plagiogrammaceae, a poorly described family of diatoms, are common inhabitants of the shallow marine littoral zone, occurring either in the sediments or as epiphytes. Previous molecular phylogenies of the Plagiogrammaceae were inferred but included only up to six genera: Plagiogramma, Dimeregramma, Neofragilaria, Talaroneis, Psammogramma and Psammoneis. In this paper, we describe a new plagiogrammoid genus, Orizaformis, obtained from Bohai Sea (China) and present molecular phylogenies of the family based on three and four genes (nuclear-encoded large and small subunit ribosomal RNAs and chloroplast-encoded rbcL and psbC). Also included in the new phylogenies is Glyphodesmis. The phylogenies suggest that the Plagiogrammaceae is composed of two major clades: one consisting of Talaroneis, Orizaformis and Psammoneis, and the second of Glyphodesmis, Psammogramma, Neofragilaria, Dimeregramma and Plagiogramma. In addition, we describe three new species within established genera: Psammoneis obaidii, which was collected from the Red Sea, Saudi Arabia; and Neofragilaria stilus and Talaroneis biacutifrons from the Mozambique Channel, Indian Ocean, and illustrate two new combination taxa: Neofragilaria anomala and Neofragilaria lineata. Our observations suggest that the biodiversity of the family is strongly needed to be researched, and the phylogenetic analyses provide a useful framework for future studies of Plagiogrammaceae.     

Feasibility of transferring fluorescent in situ hybridization probes to an 18S rRNA gene phylochip and mapping of signal intensities

DNA microarray technology offers the possibility to analyze microbial communities without cultivation, thus benefiting biodiversity studies. We developed a DNA phylochip to assess phytoplankton diversity and transferred 18S rRNA probes from dot blot or fluorescent in situ hybridization (FISH) analyses to a microarray format. Similar studies with 16S rRNA probes have been done determined that in order to achieve a signal on the microarray, the 16S rRNA molecule had to be fragmented, or PCR amplicons had to be <150 bp in length to minimize the formation of a secondary structure in the molecule so that the probe could bind to the target site. We found different results with the 18S rRNA molecule. Four out of 12 FISH probes exhibited false-negative signals on the microarray; eight exhibited strong but variable signals using full-length 18S RNA molecules. A systematic investigation of the probe's accessibility to the 18S rRNA gene was made using Prymenisum parvum as the target. Fourteen additional probes identical to this target covered the regions not tested with existing FISH probes. Probes with a binding site in the first 900 bp of the gene generated positive signals. Six out of nine probes binding in the last 900 bp of the gene produced no signal. Our results suggest that although secondary structure affected probe binding, the effect is not the same for the 18S rRNA gene and the 16S rRNA gene. For the 16S rRNA gene, the secondary structure is stronger in the first half of the molecule, whereas in the 18S rRNA gene, the last half of the molecule is critical. Probe-binding sites within 18S rRNA gene molecules are important for the probe design for DNA phylochips because signal intensity appears to be correlated with the secondary structure at the binding site in this molecule. If probes are designed from the first half of the 18S rRNA molecule, then full-length 18S rRNA molecules can be used in the hybridization on the chip, avoiding the fragmentation and the necessity for the short PCR amplicons that are associated with using the 16S rRNA molecule. Thus, the 18S rRNA molecule is a more attractive molecule for use in environmental studies where some level of quantification is desired. Target size was a minor problem, whereas for 16S rRNA molecules target size rather than probe site was important.     

Electrochemical detection of the toxic dinoflagellate Alexandrium ostenfeldii with a DNA-biosensor

The steady rise of observations of harmful or toxic algal blooms throughout the world in the past decades constitute a menace for coastal ecosystems and human interests. As a consequence, a number of programs have been launched to monitor the occurrence of harmful and toxic algae. However, the identification is currently done by microscopic examination, which requires a broad taxonomic knowledge, expensive equipment and is very time consuming. In order to facilitate the identification of toxic algae, an inexpensive and easy-to-handle DNA-biosensor has been adapted for the electrochemical detection of the toxic dinoflagellate Alexandrium ostenfeldii. The detection of the toxic algae is based on a sandwich hybridisation, which is carried out on a disposable sensor chip. A set of two probes for the species-specific identification of A. ostenfeldii was developed. The specificity of the probes could be shown in dot-blot hybridisations and with the DNA-biosensor. The sensitivity of the DNA-biosensor was optimised with respect to hybridisation temperature and NaCl-concentration and a significant increase of the sensitivity of the DNA-biosensor could be obtained by a fragmentation of the rRNA prior to the hybridisation and by adding a helper oligonucleotide, which binds in close proximity to the probes to the hybridisation.     

Ribosomal DNA sequence variation among sympatric strains of the Cyclotella meneghiniana complex (Bacillariophyceae) reveals cryptic diversity

Cyclotella meneghiniana Kützing is one of the most commonly found and intensively studied freshwater diatom species. However, it is considered taxonomically problematic because of its unusually wide ecological range and large frustule ultrastructural variation. As part of a study of morphological and genetic variation in this morphospecies, we surveyed nucleotide variation in the hypervariable D1/D2 regions of the 28S rDNA, in the ribosomal internal transcribed spacer region (containing ITS1, the 5.8S rDNA and ITS2) and in the 18S rDNA in a collection of 20 sympatric strains. High genetic variability and strong indications of genetic structure among the Cyclotella meneghiniana strains were found. Representatives of four genetically distinct--apparently reproductively isolated--groups were revealed among them. The random distribution of ITS variation within these four groups indicated that the genetic structure in Cyclotella meneghiniana can probably be explained by the presence of cryptic sexual species rather than by the lack of allogamous sexual reproduction. The morphological features traditionally used for species identification in this group cannot distinguish these putative cryptic species.     

Colorimetric detection of the toxic dinoflagellate Alexandrium minutum using sandwich hybridization in a microtiter plate assay

Rapid and reliable detection of harmful algae in coastal areas and shellfish farms is an important requirement for monitoring programs. Molecular technologies are rapidly improving the detection of phytoplankton and their toxins. Assays are based on the discrimination of genetic differences in the species. A commercially available PCR ELISA Dig Detection Kit in a microtiter plate was adapted for the rapid assessment of specificity of the two probes used in a sandwich hybridization assay. The toxic dinoflagellate Alexandrium minutum was used as the target organism and a capture and signal probe were designed for a species-specific identification of this species. This assay also provided the necessary specificity tests prior to the probes being adapted to an automated biosensor using a sandwich hybridization format. All probes regardless of the detection method must be extensively tested prior to use in the field. Total rRNA was isolated from three different strains of A. minutum and the mean concentration of RNA per cell of was determined to be 0.028 ng ± 0.003. Thus, a standard calibration curve for different RNA concentrations was determined so that cell numbers could be inferred from the assay. The assay and the standard curve were evaluated by using spiked field samples. The results demonstrated that the molecular assay was able to detect A. minutum cells at different cell counts in the presence of a complex background.     

phylochipanalyser - a program for analysing hierarchical probe sets

The recent introduction of phylochips that contain molecular probes facilitates environmental microbial identification in a single experiment without previous cultivation. A set of probes recognizing species at different taxonomic levels is denoted as a hierarchical set. Application of hierarchical probe sets on a DNA microarray allows the assessment of biodiversity with different resolutions. It significantly increases the robustness of the results retrieved from phylochip experiments because of the possible consistency checks of hybridization across different taxonomic levels. Here, we present a computer program, phylo-chipanalyser, for the hierarchy editing and the evaluation of phylochip data generated from hierarchical probe sets.