Quijano-Scheggia S., Garcés E., Lundholm N., Moestrup Ø., Andree K. and Camp J. 2009. Morphology, physiology, molecular phylogeny and sexual compatibility of the cryptic Pseudo-nitzschia delicatiss...

The frequency and intensity ofPseudo-nitzschiaspp. blooms along the coast of Catalonia have been increasing over the past 20 years. As species from this genus that are documented as toxigenic have been found in local waters, with both toxic and nontoxic species cooccurring in the same bloom, there is a need to develop management tools for discriminating the difference. Currently, differentiation of toxic and nontoxic species requires time-consuming electron microscopy to distinguish taxonomic features that would allow identification as to species, and cryptic species can still remain misidentified. In this study, cells ofPseudo-nitzschiafrom clonal cultures isolated from seawater were characterized to their species identity using scanning electron microscopy, and subsamples of each culture were used to create an internal transcribed spacer 1 (ITS-1), 5.8S, and ITS-2 ribosomal DNA database for development of species-specific quantitative PCR (qPCR) assays. Once developed, these qPCR assays were applied to field samples collected over a 2-year period in Alfaques Bay in the northwestern Mediterranean Sea to evaluate the possibility of a comprehensive surveillance for allPseudo-nitzschiaspp. using molecular methods to supplement optical microscopy, which can discern taxonomy only to the genus level within this taxon. TotalPseudo-nitzschiacell density was determined by optical microscopy from water samples collected weekly and compared to results obtained from the sum of eightPseudo-nitzschiaspecies-specific qPCR assays using duplicate samples. Species-specific qPCR followed by melt curve analysis allowed differentiation of amplicons and identification of false positives, and results correlated well with the totalPseudo-nitzschiacell counts from optical microscopy.

Proliferations of the genus Pseudo-nitzschia recur along the Catalan coast (NW Mediterranean) throughout the year. The establishment of 58 clonal cultures facilitated morphological studies with scanning electron microscopy (SEM) and ITS 5.8S rDNA sequence characterization. Moreover, strains of each species were examined with respect to sexual compatibility and toxicity. The results of the morphological and phylogenetic studies confirmed nine species of the genus Pseudo-nitzschia: P. brasiliana, P. calliantha, P. delicatissima clade A/del 2, P. arenysensis, P. fraudulenta, P. galaxiae, P. linea, P. multistriata and P. pungens clade I. Moreover, two Pseudo-nitzschia species, P. caciantha and P. cf mannii, could only be identified following SEM analysis of their morphology. None of the cultured strains of Pseudo-nitzschia analyzed produced domoic acid in amounts above the limit of detection. The current distributions of the Pseudo-nitzschia species in the Mediterranean Sea were plotted on maps, which led to the following observations: i) diversity within this genus is much greater than previously considered, ii) some species have a broad distribution (e.g. P. calliantha), iii) whereas the distribution of others is narrowly restricted (e.g. P. pungens clade I). Moreover, this study reports the first detection of P. linea in the Mediterranean Sea and is the first description of P. galaxiae and P. cf mannii along the Catalan coast. Morphological studies coupled with molecular biological characterization, mating tests and biogeographic distribution analyses provide a critical theoretical basis for testing and/or implementing the current phylogenetic framework in the genus Pseudo-nitzschia.