Phylogeny and species delineation in the marine diatom Pseudo-nitzschia (Bacillariophyta) using cox1, LSU, and ITS2 rRNA genes: A perspective in character evolution.

Analyses of the mitochondrial cox1, the nuclear-encoded large subunit (LSU), and the internal transcribed spacer 2 (ITS2) RNA coding region of Pseudo-nitzschia revealed that the P. pseudodelicatissima complex can be phylogenetically grouped into three distinct clades (Groups I-III), while the P. delicatissima complex forms another distinct clade (Group IV) in both the LSU and ITS2 phylogenetic trees. It was elucidated that comprehensive taxon sampling (sampling of sequences), selection of appropriate target genes and outgroup, and alignment strategies influenced the phylogenetic accuracy. Based on the genetic divergence, ITS2 resulted in the most resolved trees, followed by cox1 and LSU. The morphological characters available for Pseudo-nitzschia, although limited in number, were overall in agreement with the phylogenies when mapped onto the ITS2 tree. Information on the presence/absence of a central nodule, number of rows of poroids in each stria, and of sectors dividing the poroids mapped onto the ITS2 tree revealed the evolution of the recently diverged species. The morphologically based species complexes showed evolutionary relevance in agreement with molecular phylogeny inferred from ITS2 sequence-structure data. The data set of the hypervariable region of ITS2 improved the phylogenetic inference compared to the cox1 and LSU data sets. The taxonomic status of P. cuspidata and P. pseudodelicatissima requires further elucidation.

Characterization of a Strain of Fukuyoa paulensis (Dinophyceae) from the Western Mediterranean Sea.

A single cell of the dinoflagellate genus Fukuyoa was isolated from the island of Formentera (Balearic Islands, west Mediterranean Sea), cultured, and characterized by morphological and molecular methods and toxin analyses. This is the first report of the Gambierdiscus lineage (genera Fukuyoa and Gambierdiscus) from the western Mediterranean Sea, which is cooler than its eastern basin. Molecular analyses revealed that the Mediterranean strain belongs to F. paulensis and that it bears LSU rDNA sequences identical to New Zealand, Australian, and Brazilian strains. It also shared an identical sequence of the more variable ITS-rDNA with the Brazilian strain. Toxin analyses showed the presence of maitotoxin, 54-deoxyCTX1B, and gambieric acid A. This is the first observation of the two latter compounds in a Fukuyoa strain. Therefore, both Gambierdiscus and Fukuyoa should be considered when as contributing to ciguatera fish poisoning. Different strains of Fukuyoa form a complex of morphologically cryptic lineages where F. paulensis stands as the most distantly related nominal species. The comparison of the ITS2 secondary structures revealed the absence of CBCs among strains. The study of the morphological and molecular traits depicted an unresolved taxonomic scenario impacted by the low strains sampling.

Characterization of Prorocentrum elegans and Prorocentrum levis (Dinophyceae) from the southeastern Bay of Biscay by morphology and molecular phylogeny.

Benthic Prorocentrum species can produce toxins that adversely affect animals and human health. They are known to co-occur with other bloom-forming, potentially toxic, benthic dinoflagellates of the genera Ostreopsis, Coolia, and Gambierdiscus. In this study, we report on the presence of P. elegans M.Faust and P. levis M.A.Faust, Kibler, Vandersea, P.A. Tester & Litaker from the southeastern Bay of Biscay. Sampling was carried out in the Summer-Autumn 2010-2012 along the Atlantic coast of the Iberian Peninsula, but these two species were only found in the northeastern part of the Peninsula. Strains were isolated from macroalgae collected from rocky-shore areas bordering accessible beaches. Morphological traits of isolated strains were analyzed by LM and SEM, whereas molecular analyses were performed using the LSU and internal transcribed spacer (ITS)1-5.8S-ITS2 regions of the rDNA. A bioassay with Artemia fransciscana and liquid chromatography-high-resolution mass spectrometry analyses were used to check the toxicity of the species, whose results were negative. The strains mostly corresponded to their species original morphological characterization, which is supported by the phylogenetic analyses in the case of P. levis, whereas for P. elegans, this is the first known molecular characterization. This is also the second known report of P. elegans.

Physiological response of Prorocentrum lima (Dinophyceae) to varying light intensities.

The benthic dinoflagellate Prorocentrum lima is among the most common toxic morphospecies with a cosmopolitan distribution. This study explored if strains from different environments and different morphotypes, isolated from three locations in the Atlantic Iberian Peninsula and two from the Mediterranean Sea, showed different responses to varying light regimes, after confirming that all strains belonged to the same ribotype. Growth rates and photosynthetic parameters such as Fo, Fv/Fm, and rETRmax were analysed with a Coulter counter, a water-PAM and a fast repetition rate fluorometer. The photosynthetic properties were investigated in a high light stress experiment using strains acclimated to low light (LL) and high light (HL). The highest growth rate was 0.23 day-1 at 80 and 100 µmol photons m-2 s-1 for strains Dn150EHU and Dn60EHU, originated from different locations. Under control conditions (18°C and 90 µmol photons m-2 s-1), growth rate was on average 0.10 day-1. The HL stress exposure induced photodamage to all strains and the recovery period was not sufficiently long for full recovery of Fv/Fm. However, cells acclimated to HL showed a better recovery than the LL acclimated ones. Furthermore, some assumptions are discussed in relation to strains' original location.

The genus Pseudo-nitzschia (Bacillariophyceae) in a temperate estuary with description of two new species: Pseudo-nitzschia plurisecta sp. nov. and Pseudo-nitzschia abrensis sp. nov.

The genus Pseudo-nitzschia contains potentially toxic species of problematic taxonomy, making it one of the most intensively studied diatom genera. The study of 35 clonal strains isolated from the Bilbao estuary, an area that experiences recurrent blooms of Pseudo-nitzschia, revealed the presence of two new species, P. abrensis and P. plurisecta, differing from their congeners in both morphology and gene sequence. The morphological features were analyzed by LM and EM, whereas molecular analyses were based on the internal transcribed spacer (ITS) and large subunit (LSU) regions of the rDNA. P. plurisecta appears closely related to P. cuspidata/P. pseudodelicatissima in the phylogenetic tree, whereas P. abrensis forms a moderately supported clade with P. heimii/P. subpacifica and P. caciantha/P. circumpora. Comparison of the secondary structure of ITS2 regions reveals marked differences in the most highly conserved regions among related taxa. Morphologically, the new species differ from their closest congeners in the arrangement of the poroid sectors and the density of valve striae and fibulae. The two species share similar pigment composition, and belong to the group of Pseudo-nitzschia species containing only chlorophyll c2 and c3 .

Potential risk of harmful algae transport by ballast waters: the case of Bilbao Harbour.

Ballast water exchange was measured for the first time in Bilbao Harbour, one of the most active of northern Spain. Between 1997 and 2006, 41,900,980.34 ballast water tn were loaded and 13,272,709 tn were discharged. Bilbao Harbour appears to be mainly a source of ballast water, 90% of which would be discharged in European harbours. We estimated that vessels carrying liquid and solid bulk have higher probabilities of exporting ballast water, whereas those with liquid bulk and containers are more likely to introduce it. From 30 potentially harmful phytoplankton species identified to date near harbour facilities, there would be a high risk of exporting at least Alexandrium minutum, Dinophysis sp., Heterosigma akashiwo, Karlodinium sp., Ostreopsis cf. siamensis, Pfiesteria-like and Prorocentrum minimum. Invasion risk by ballast water was tested by analyzing the response of six strains of H. akashiwo from different geographic areas to varying salinity. Results show that successful growth of foreign strains would be possible.

Phytoplankton pigments and epifluorescence microscopy as tools for ecological status assessment in coastal and estuarine waters, within the Water Framework Directive.

Inverted microscopy is widespread employed for the analysis of phytoplankton composition within water quality monitoring networks. However, the analysis at the lowest taxonomical level is not always required for ecological status assessment. In addition, inverted microscopy can underestimate the small phytoplankton, and not always distinguish photoautotrophic from heterotrophic cells. In this study, as alternative tools, epifluorescence microscopy and High Performance Liquid Chromatography (HPLC) were employed to characterize phytoplankton communities within waters of different trophic condition. Epifluorescence microscopy confirmed its effectiveness to count the small phytoplankton. Furthermore, significant correlations between nutrients of anthropogenic origin and nanoplankton abundances were found. However, this technique resulted very time-consuming. HPLC together with the CHEMTAX program was more appropriate than inverted microscopy, in terms of cost-effectiveness. Also, the main variability patterns observed in the phytoplankton community structure by HPLC coincided with previous findings in the study area. Nevertheless, a rapid screening at the inverted microscope is recommended.