Jordi Camp

Morphology, physiology, molecular phylogeny and sexual compatibility of the cryptic Pseudo-nitzschia delicatissima complex (Bacillariophyta), including the description of P. arenysensis sp. nov.

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 delicatissima complex (Bacillariophyta), including the description of P. arenysensis sp. nov. Phycologia 48: 492–509. DOI: 10.2216/08-21.1 Several strains of Pseudo-nitzschia delicatissima were isolated from the northwestern Mediterranean Sea and compared using light and electron microscopy, phylogenetic analyses of internal transcribed spacer rDNA, together with studies of their mating system, estimations of growth rates and reduction in cell size with time. Morphological and phylogenetic analyses identified all strains as Pseudo-nitzschia delicatissima, but in the phylogenetic analyses they fell into two genetically distinct clades. Studies of fine structural morphological characteristics by electron microscopy did not allow discrimination of the two clades. Mating experiments showed successful sexualisation among strains of clade B/1; whereas, mating attempts in clade A/2 and between clade A/2 and clade B/1 were unsuccessful under the examined laboratory conditions. The morphological characteristics of the daughter generations F1 and F2 were analyzed and abnormalities in the F2 generation described. We describe for the first time the rapid size reduction in the F2 generation. Culture studies showed physiological differences between the two clades; strains from clade A/2 showed a tendency for higher growth rates and slower reduction in cell size than strains from clade B/1. Morphological and phylogenetic studies comparing strains from different parts of the world showed P. delicatissima as a cryptic species complex comprising at least two different genotypes, both apparently cosmopolitan. The two genotypes were shown to occur sympatrically in the Mediterranean Sea, and we hypothesize that the genotypes were most likely separated by reproductive barriers associated with sexualisation occurring at different temperatures and hence different seasons. The molecular and physiological differences allow us to describe one of the genotypes, clade B/1, as a new species, Pseudo-nitzschia arenysensis sp. nov. Differences in growth capabilities of the cultures were observed in relation to cell size and physiological status and showed a nonlinear relationship between cell size and growth. Possibly, such capability plays a key role in the occurrence and dynamics of phytoplankton algae.

LIFE HISTORY AND IN SITU GROWTH RATES OF ALEXANDRIUM TAYLORI (DINOPHYCEAE, PYRROPHYTA)

Alexandrium taylori Balech is a phototrophic marine dinoflagellate. It produced recurrent blooms during the summer months (July and August) of 1994 to 1997 in La Fosca beach (NW Mediterranean). In addition to a motile vegetative form, A. taylori had two benthic forms: temporary cysts and resting cysts. Temporary cysts were a temporally quiescent stage produced from the ecdysis of the vegetative cell in both natural populations and laboratory cultures. Temporary cysts may divide to form motile cells. Resting cysts had a thicker wall than the temporary cysts and had a red accumulation body. Gametes and planozygotes were also observed in laboratory cultures. Alexandrium taylori showed in situ diurnal vertical migration with an increase of vegetative cells in the water column in the morning through midday, with concentrations peaking in the afternoon followed by lower levels at night. Most vegetative cells lost their thecae and flagella, and with them their motility, turning into temporary cysts that settled in the early evening. The number of temporary cysts in the water column rose in the evening and at night. The temporary cysts gave rise to motile cells the following morning. Synthesis of DNA occurred in vegetative cells at night, and a preferential period of cell division occurred at sunrise. The estimated division rate in the field was 0.4–0.5 vegetative cells·day−1. Temporary cysts had twice the DNA of a G1 vegetative cell. The minimum in situ division rate of the temporary cysts was 0.14 day−1. The role of the resting and temporary cyst population in the annual recurrence and maintenance of the A. taylori bloom is discussed.

Characterization of NW Mediterranean Karlodinium spp. (Dinophyceae) strains using morphological, molecular, chemical, and physiological methodologies

Recurrent fish kills in the Spanish Alfacs Bay (NW Mediterranean) have been detected during winter seasons since 1994, and were attributed to an unarmored, ichthyotoxic, dinoflagellate, initially identified as Gyrodinium corsicum Paulmier, Berland, Billard, & Nezan. Several strains were isolated from the bay and their clonal cultures were compared by combined techniques, including light and electron microscopy, internal transcribed spacer and 5.8S rDNA nucleotide sequencing, and HPLC pigment analyses, together with studies of their photochemical performance, growth rates, and toxicity. Using phylogenetic analyses, all strains were identified as members of the genus Karlodinium, but they were separated into two genetically distinct groups. These groups, identified as Karlodinium veneficum (Ballantine) J. Larsen and K. armiger Bergholtz, Daugbjerg et. Moestrup, were also supported by the other techniques used. Detailed analyses of fine structural characteristics (including plug‐like structures in amphiesma and a possible layer of semi‐opaque material beneath the outer membrane) allowed discrimination of the mentioned two species. Specific differences in pigment patterns coincided with that expected for low‐ (K. veneficum) and high‐light (K. armiger) adapted relatives. The higher photosynthetic efficiency of K. veneficum and the longer reactivation times of the PSII reaction centers observed for K. armiger were in agreement with this hypothesis. The two species differed in toxicity, but the strains used always induced mortality when incubated with bivalves, rotifers, and finfish. Compared with K. armiger, strains of K. veneficum yielded higher cell densities, but had lower growth rates.

Quantitative PCR Coupled with Melt Curve Analysis for Detection of Selected Pseudo-nitzschia spp. (Bacillariophyceae) from the Northwestern Mediterranean Sea

ABSTRACT The frequency and intensity of Pseudo-nitzschia spp. 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 of Pseudo-nitzschia from 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 all Pseudo-nitzschia spp. using molecular methods to supplement optical microscopy, which can discern taxonomy only to the genus level within this taxon. Total Pseudo-nitzschia cell density was determined by optical microscopy from water samples collected weekly and compared to results obtained from the sum of eight Pseudo-nitzschia species-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 total Pseudo-nitzschia cell counts from optical microscopy.

Description, Host-specificity, and Strain Selectivity of the Dinoflagellate Parasite Parvilucifera sinerae sp. nov. (Perkinsozoa)

A new species of parasite, Parvilucifera sinerae sp. nov., isolated from a bloom of the toxic dinoflagellate Alexandrium minutum in the harbor of Arenys de Mar (Mediterranean Sea, Spain), is described. This species is morphologically, behaviourally, and genetically (18S rDNA sequence) different from Parvilucifera infectans, until now the only species of the genus Parvilucifera to be genetically analyzed. Sequence analysis of the 18S ribosomal DNA supported P. sinerae as a new species placed within the Perkinsozoa and close to P. infectans. Data on the seasonal occurrence of P. sinerae, its infective rates in natural and laboratory cultures, and intra-species strain-specific resistance are presented. Life-cycle studies in field samples showed that the dinoflagellate resting zygote (resting cyst) was resistant to infection, but the mobile zygote (planozygote) or pellicle stage (temporary cyst) became infected. The effects of light and salinity levels on the growth of P. sinerae were examined, and the results showed that low salinity levels promote both sporangial germination and higher rates of infection. Our findings on this newly described parasite point to a complex host-parasite interaction and provide valuable information that leads to a reconsideration of the biological strategy to control dinoflagellate blooms by means of intentional parasitic infections.

BARRUFETA BRAVENSIS GEN. NOV. SP. NOV. (DINOPHYCEAE): A NEW BLOOM-FORMING SPECIES FROM THE NORTHWEST MEDITERRANEAN SEA(1).

The present study describes a new dinoflagellate genus, Barrufeta N. Sampedro et S. Fraga gen. nov., with one new species, B. bravensis Sampedro et S. Fraga sp. nov., isolated from the Costa Brava (NW Mediterranean Sea). The dinoflagellate was characterized at the genus and species levels by LM and EM; LSU and internal transcribed spacer (ITS) rDNA sequences; and HPLC analyses of the pigments, fatty acids, and possible presence of toxins of several cultured strains. The new Barrufeta species is oval shaped (22–35 μm long and 16–25 μm wide) and dorsoventrally flattened. It possesses numerous small chloroplasts that radiate from two large equatorially located pyrenoids and is a typical peridinin‐containing dinoflagellate. The nucleus is in the anterior part of the epicone. The apical groove has a characteristic “Smurf‐cap” shape that runs counterclockwise on the epicone and terminates on its right posterior part. B. bravensis is similar to the previously described species Gyrodinium resplendens Hulburt in its external morphology, but the original report of the latter lacked a description of the complete shape of the apical groove. It is therefore likely that some of the G. resplendens species reported in the literature are Barrufeta since they possess a Barrufeta‐type apical groove. Fatty acids of Barrufeta were more similar to those of Karenia brevis than those obtained from other unarmored analyzed species including three species of Gymnodinium and Akashiwo sanguinea.

Short-term variations in development of a recurrent toxic Alexandrium minutum–dominated dinoflagellate bloom induced by meteorological conditions1

Development of an Alexandrium minutum Halim bloom affecting a Mediterranean harbor was monitored in detail using a multidisciplinary approach. A. minutum was by far the most abundant species at and near the bloom maximum, but always coexisted with members of three additional dinoflagellate genera and prasinophytes. Bloom initiation (early February) occurred during prolonged influences of sunny weather conditions, when day length exceeded 10.5 h and water temperatures reached 10.2°C. Subsequent development toward its maximum (end of March) also relied on good weather conditions, with specific wind directions favoring accumulation of cells. Arrival of rainy weather, associated with frontal boundaries of large‐scale low‐atmospheric‐pressure systems and characterized by reduced solar irradiance (heavy cloud coverage), opposite wind directions, and enhanced wind speeds, always caused temporal declines of the bloom. These declines were attributed to dispersal or displacement of algae, but a vertical migration of A. minutum cells toward the sediment was not excluded. Delayed inflows of excess terrestrial rainwater along the inner harbor wall strongly reduced salinity and prolonged a temporal decline far beyond influences of bad weather. The associated nutrient supply favored development of the phytoplankton population but reduced the toxin production of A. minutum cells. The HPLC‐determined Gonyautoxin (GTX) 1 + 4/GTX 2 + 3 ratio strongly increased toward the bloom maximum. This ratio was influenced by nutrient status and cell density and has a potential value for monitoring developmental stages of blooms. Prolonged bad weather conditions eventually hindered continuation of bloom development, and subsequent declines of algal biomass were attributed to grazing.

Management of Ostreopsis Blooms in Recreational waters along the Catalan Coast (NW Mediterranean Sea): Cooperation between a Research Project and a Monitoring Program

Abstract As shown in this report, the integration of a research project with a monitoring program improves the detection and management of Ostreopsis blooms in Catalonia. The research project benefits from information previously obtained from several localities by the monitoring program, which in turn profits from the specific findings and conclusions contributed by the research project.

HOMOTHALLIC AUXOSPORULATION IN PSEUDO-NITZSCHIA BRASILIANA (BACILLARIOPHYTA)1

Most pennate diatoms are allogamous, and various types of mating systems have been described. In Pseudo‐nitzschia, reproductive stages have been identified in some species, and it is generally accepted that the genus is mainly heterothallic. Here we report homothallic auxosporulation of Pseudo‐nitzschia brasiliana Lundholm, Hasle et G. A. Fryxell. To our knowledge, this is the first verified description of homothallic sexual reproduction in the genus. Auxospore formation was observed in all 16 subclones derived from three initial clonal cultures of P. brasiliana. Pairing was followed by production of two gametes per gametangium, which fused to give two zygotes. Each zygote (early auxospore) was initially spherical and adhered to one girdle band of the parental frustule. The two auxospores tended to expand parallel to each other and perpendicular to the parental frustule. Elongation was synchronous, slightly asynchronous, or totally asynchronous. The entire process of sexual reproduction, from gamete formation to the appearance of the initial vegetative cells, took 2–4 d. The occurrence of sex in a homothallic species seems an advantageous life strategy for this species in that any encounter between cells of the right size class is potentially sexual.

Diversity and Phylogeny of Gymnodiniales (Dinophyceae) from the NW Mediterranean Sea Revealed by a Morphological and Molecular Approach.

The diversity and phylogeny of dinoflagellates belonging to the Gymnodiniales were studied during a 3-year period at several coastal stations along the Catalan coast (NW Mediterranean) by combining analyses of their morphological features with rDNA sequencing. This approach resulted in the detection of 59 different morphospecies, 13 of which were observed for the first time in the Mediterranean Sea. Fifteen of the detected species were HAB producers; four represented novel detections on the Catalan coast and two in the Mediterranean Sea. Partial rDNA sequences were obtained for 50 different morphospecies, including novel LSU rDNA sequences for 27 species, highlighting the current scarcity of molecular information for this group of dinoflagellates. The combination of morphology and genetics allowed the first determinations of the phylogenetic position of several genera, i.e., Torodinium and many Gyrodinium and Warnowiacean species. The results also suggested that among the specimens belonging to the genera Gymnodinium, Apicoporus, and Cochlodinium were those representing as yet undescribed species. Furthermore, the phylogenetic data suggested taxonomic incongruences for some species, i.e., Gyrodinium undulans and Gymnodinium agaricoides. Although a species complex related to G. spirale was detected, the partial LSU rDNA sequences lacked sufficient resolution to discriminate between various other Gyrodinium morphospecies.