Interspecific plastidial recombination in the diatom genus Pseudo‐nitzschia

Plastids are usually uni‐parentally inherited and genetic recombination between these organelles is seldom observed. The genus Pseudo‐nitzschia, a globally relevant marine diatom, features bi‐parental plastid inheritance in the course of sexual reproduction. This observation inspired the recombination detection we pursued in this paper over a ~1,400‐nucleotide‐long region of the plastidial rbcL, a marker used in both molecular taxonomy and phylogenetic studies in diatoms. Among all the rbcL‐sequences available in web‐databases for Pseudo‐nitzschia, 42 haplotypes were identified and grouped in five clusters by Bayesian phylogeny. Signs of hybridization were evident in four of five clusters, at both intra‐ and interspecific levels, suggesting that, in diatoms, (i) plastidial recombination is not absent and (ii) hybridization can play a role in speciation of Pseudo‐nitzschia spp.     

The dynamics of sexual phase in the marine diatom Pseudo‐nitzschia multistriata (Bacillariophyceae)

Sexual reproduction represents a fundamental phase in the life cycle of diatoms, linked to both the production of genotypic diversity and the formation of large‐sized initial cells. Only cells below a certain size threshold can be sexualized, but various environmental factors can modulate the success of sexual reproduction. We investigated the role of cell density and physiological conditions of parental strains in affecting the success and timing of sexual reproduction in the marine heterothallic diatom Pseudo‐nitzschia multistriata. We also studied the dynamics of the sexual phase in still conditions allowing cell sedimentation and in gently mixed conditions that keep cells in suspension. Our results showed that successful sexual reproduction can only be achieved when crossing parental strains in the exponential growth phase. Evidence was provided for the fact that sexual reproduction is a density‐dependent event and requires a threshold cell concentration to start, although this might vary considerably amongst strains. Moreover, the onset of the sexual phase was coupled to a marked reduction in growth of the vegetative parental cells. The crosses carried out in physically mixed conditions produced a significantly reduced number of sexual stages as compared to crosses in still conditions, showing that mixing impairs sexualization. The results of our experiments suggest that the signaling that triggers the sexual phase is favored when cells can accumulate, reducing the distance between them and facilitating contacts and/or the perception of chemical cues. Information on the progression of the sexual phase in laboratory conditions help understanding the conditions at which sex occurs in the natural environment.     

Influence of sudden salinity variation on the physiology and domoic acid production by two strains of Pseudo‐nitzschia australis

Several coastal countries including France have experienced serious and increasing problems related to Pseudo‐nitzschia toxic blooms. These toxic blooms occur in estuarine and coastal waters potentially subject to fluctuations in salinity. In this study, we document for the first time the viability, growth, photosynthetic efficiency, and toxin production of two strains of Pseudo‐nitzschia australis grown under conditions with sudden salinity changes. Following salinity variation, the two strains survived over a restricted salinity range of 30–35, with favorable physiological responses, as the growth, effective quantum yield and toxin content were high compared to the other conditions. In addition, high cellular quotas of domoic acid (DA) were observed at a salinity of 40 for the strain IFR‐PAU‐16.1 in comparison with the other strain IFR‐PAU‐16.2 where the cell DA content was directly released into the medium. On the other hand, the osmotic stress imposed at lower salinities, 20 and 10, resulted in cell lysis and a sudden DA leakage in the medium. Intra‐specific variability was observed in growth and toxin production, with the strain IFR‐PAU‐16.1 apparently able to withstand higher salinities than the strain IFR‐PAU‐16.2. On the whole, DA does not appear to act as an osmolyte in response to sudden salinity changes. Since most of the shellfish harvesting areas of bivalve molluscs in France are located in areas where the salinity generally varies between 30 and 35, Pseudo‐nitzschia australis blooms might potentially impact public health and commercial shellfish resources in these places.     

The first report of the potentially harmful diatom Pseudo‐nitzschia caciantha from Australian coastal waters

The diatom Pseudo‐nitzschia is a significant component of coastal waters worldwide and a producer of the potent neurotoxin, domoic acid. Sixteen species belonging to this genus have been reported from Australian waters, but the potentially toxic species P. caciantha has not been previously known from this region. Two clonal strains of P. caciantha were isolated from Coogee Beach, south‐east Australia, and the morphological, molecular and toxicological evidence for this species delineation were examined using light and transmission electron microscopy, phylogenetic analysis based on sequences of the second internal transcribed spacer and domoic acid production as measured by liquid chromatography–mass spectrometry. The results unambiguously confirmed that these isolates are the potentially toxic species P. caciantha , being only the second report of this species in the Southern Hemisphere. The potential for further hidden Pseudo‐nitzschia diversity in these waters is considerable.     

A polyphasic approach to the study of the genus Nitzschia (Bacillariophyta): three new planktonic species from the Adriatic Sea

The paraphyletic diatom genus Nitzschia comprises over 1000 morphologically distinct pennate taxa, known from the benthos and plankton of freshwater, brackish, and marine environments. The principal diagnostic characters for delimitation of Nitzschia species include valve shape, the position and structure of the raphe, presence/absence and shape of the proximal raphe endings and terminal raphe fissures, areola structure, and specific morphometric features such as cell size, and stria and fibula density. In this study, we isolated 12 diatom strains into culture from samples collected at the surface or greater depths of the southeastern Adriatic Sea. Morphological analyses included LM, SEM, and TEM observations, which, along with specific morphometric features, allowed us to distinguish three new Nitzschia species. These findings were congruent with the results of phylogenetic analyses performed on nuclear‐encoded SSU (18S) rDNA and chloroplast‐encoded rbcL and psbC genes. One of the new species (Nitzschia dalmatica sp. nov.) formed a lineage within a clade of Bacillariaceae containing members of the Nitzschia sect. Dubiae, which was sister to Psammodictyon. A second lineage was part of a novel clade that is significantly distinct from other Nitzschia species sequenced so far and includes Nitzschia adhaerens sp. nov. and N. cf. adhaerens. A further new species was found, Nitzschia inordinata sp. nov., which appeared as the sister group to the N. adhaerens clade and the conopeoid Nitzschia species in our phylogenetic trees. Our findings contribute to the overall diversity of genus Nitzschia, especially in identifying some deep branches within the Bacillariaceae, and highlight under‐scoring of this genus in marine plankton.     

Intraspecific diversity and distribution of the cosmopolitan species Pseudo‐nitzschia pungens (Bacillariophyceae): morphology,genetics, and ecophysiology of the three clades

Three clades of Pseudo‐nitzschia pungens, determined by the internal transcribed space (ITS) region, are distributed throughout the world. We studied 15 P. pungens clones from various geographical locations and confirmed the existence of the three clades within P. pungens, based on ITS sequencing and described the three subgroups (IIIaa, IIIab, and IIIb) of clade III. Clade III (clade IIIaa) populations were reported for the first time in Korean coastal waters and the East China Sea. In morphometric analysis, we found the ultrastructural differences in the number of fibulae, striae, and poroids that separate the three clades. We carried out physiological tests on nine clones belonging to the three clades growing under various culture conditions. In temperature tests, only clade III clones could not grow at lower temperatures (10°C and 15°C), although clade I and II clones grew well. The estimated optimal growth range of clade I clones was wider than that of clades II and III. Clade II clones were considered to be adapted to lower temperatures and clade III to higher temperatures. In salinity tests, clade II and III clones did not grow well at a salinity of 40. Clade I clones were regarded as euryhaline and clade II and III clones were stenohaline. This supports the hypothesis that P. pungens clades have different ecophysiological characteristics based on their habitats. Our data show that physiological and morphological features are correlated with genetic intraspecific differentiation in P. pungens.     

Morphological and genetic diversity of Beaufort Sea diatoms with high contributions from the Chaetoceros neogracilis species complex

Seventy‐five diatom strains isolated from the Beaufort Sea (Canadian Arctic) in the summer of 2009 were characterized by light and electron microscopy (SEM and TEM), as well as 18S and 28S rRNA gene sequencing. These strains group into 20 genotypes and 17 morphotypes and are affiliated with the genera Arcocellulus, Attheya, Chaetoceros, Cylindrotheca, Eucampia, Nitzschia, Porosira, Pseudo‐nitzschia, Shionodiscus, Thalassiosira, and Synedropsis. Most of the species have a distribution confined to the northern/polar area. Chaetoceros neogracilis and Chaetoceros gelidus were the most represented taxa. Strains of C. neogracilis were morphologically similar and shared identical 18S rRNA gene sequences, but belonged to four distinct genetic clades based on 28S rRNA, ITS‐1 and ITS‐2 phylogenies. Secondary structure prediction revealed that these four clades differ in hemi‐compensatory base changes (HCBCs) in paired positions of the ITS‐2, suggesting their inability to interbreed. Reproductively isolated C. neogracilis genotypes can thus co‐occur in summer phytoplankton communities in the Beaufort Sea. C. neogracilis generally occurred as single cells but also formed short colonies. It is phylogenetically distinct from an Antarctic species, erroneously identified in some previous studies as C. neogracilis, but named here as Chaetoceros sp. This work provides taxonomically validated sequences for 20 Arctic diatom taxa, which will facilitate future metabarcoding studies on phytoplankton in this region.     

First report of the potentially toxic marine diatom Pseudo‐nitzschia simulans (Bacillariophyceae) from the East Australian Current

Certain species of the marine diatom genus Pseudo‐nitzschia are responsible for the production of the domoic acid (DA), a neurotoxin that can bioaccumulate in the food chain and cause amnesic shellfish poisoning (ASP) in animals and humans. This study extends our knowledge by reporting on the first observation of the potentially toxic species Pseudo‐nitzschia simulans from this region. One clonal strain of P. simulans was isolated from the East Australian Current and characterized using light and transmission electron microscopy, and phylogenetic analyses based on regions of the internal transcribed spacer (ITS) and the D1–D3 region of the large subunit (LSU) of the nuclear‐encoded ribosomal deoxyribonucleic acid (rDNA), as well as examined for DA production as measured by liquid chromatography–mass spectrometry. Although this strain was non‐toxic under the defined growth conditions, the results unambiguously confirmed that this isolate is the potentially toxic species P. simulans – the first report of this species from the Southern Hemisphere.     

First reports of Pseudo‐nitzschia micropora and P. hasleana (Bacillariaceae) from the Southern Hemisphere: Morphological,molecular and toxicological characterization

Pseudo‐nitzschia H. Peragallo is a marine diatom genus found worldwide in polar, temperate, subtropical and tropical waters. It includes toxigenic representatives that produce domoic acid (DA), a neurotoxin responsible for Amnesic Shellfish Poisoning. In this study we characterized two species of Pseudo‐nitzschia collected from Port Stephens and the Hawkesbury River (south eastern Australia) previously unreported from Australian waters. Clonal isolates were sub‐sampled for (i) light and transmission electron microscopy; (ii) DNA sequencing, based on the nuclear‐encoded partial large subunit ribosomal RNA gene and internal transcribed spacer (ITS)‐ITS1, 5.8S and ITS2 rDNA regions and, (iii) DA production as measured by liquid chromatography‐mass spectrometry. Morphological and molecular data unambiguously revealed the species to be Pseudo‐nitzschia micropora Priisholm, Moestrup & Lundholm (Port Stephens) and Pseudo‐nitzschia hasleana Lundholm (Hawkesbury River). This is the first report of the occurrence of these species from the Southern Hemisphere and the first report of P. micropora in warm‐temperate waters. Cultures of P. micropora, tested for DA production for the first time, proved to be non‐toxic. Similarly, no detectable toxin concentrations were observed for P. hasleana. Species resolution and knowledge on the toxicity of local Pseudo‐nitzschia species has important implications for harmful algal bloom monitoring and management.     

Nitrogen Utilization and Toxin Production by Two Diatoms of the Pseudo‐nitzschia pseudodelicatissima Complex: P. cuspidata and P. fryxelliana

The toxigenic diatom Pseudo‐nitzschia cuspidata, isolated from the U.S. Pacific Northwest, was examined in unialgal batch cultures to evaluate domoic acid (DA) toxicity and growth as a function of light, N substrate, and growth phase. Experiments conducted at saturating (120 μmol photons · m^?2 · s^?1) and subsaturating (40 μmol photons · m^?2 · s^?1) photosynthetic photon flux density (PPFD), demonstrate that P. cuspidata grows significantly faster at the higher PPFD on all three N substrates tested [nitrate (NO₃^?), ammonium (NH₄⁺), and urea], but neither cellular toxicity nor exponential growth rates were strongly associated with one N source over the other at high PPFD. However, at the lower PPFD, the exponential growth rates were approximately halved, and the cells were significantly more toxic regardless of N substrate. Urea supported significantly faster growth rates, and cellular toxicity varied as a function of N substrate with NO₃^?‐supported cells being significantly more toxic than both NH₄⁺‐ and urea‐supported cells at the low PPFD. Kinetic uptake parameters were determined for another member of the P. pseudodelicatissima complex, P. fryxelliana. After growth of these cells on NO₃^? they exhibited maximum specific uptake rates (V_max) of 22.7, 29.9, 8.98 × 10^?3 · h^?1, half‐saturation constants (K_s) of 1.34, 2.14, 0.28 μg‐at N · L^?1, and affinity values (α) of 17.0, 14.7, 32.5 × 10^?3 · h^?1/(μg‐at N · L^?1) for NO₃^?, NH₄⁺ and urea, respectively. These labo‐ratory results demonstrate the capability of P. cuspidata to grow and produce DA on both oxidized and reduced N substrates during both exponential and stationary growth phases, and the uptake kinetic results for the pseudo‐cryptic species, P. fryxelliana suggest that reduced N sources from coastal runoff could be important for maintenance of these small pennate diatoms in U.S. west coast blooms, especially during times of low ambient N concentrations.     

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.     

Interaction of l‐leucyl‐l‐leucyl‐l‐leucine thin film with water and organic vapors: receptor properties and related morphology

The ability of highly ordered tripeptide structures to keep or change their morphology in contact with organic vapors was studied. A thin film of tripeptide l ‐leucyl‐l ‐leucyl‐l ‐leucine (LLL) was prepared having microcrystals and nanocrystals on its surface, which are stable upon vacuum drying but become objects of selective morphology change after a contact with vapors of organic solvents. Fine separate LLL crystals and their agglomerates of submicron and larger dimensions were observed by atomic force microscopy and scanning electron microscopy. After saturation with guest vapors, these crystals can remain intact or change their morphology with the increase in size or complete destruction depending on the guest molecular structure. The crystals completely lose their shape after the binding of pyridine vapors. The other studied guests produce much smaller transformations or have no effect on crystal morphology despite being sorbed by solid LLL, which was shown using quartz crystal microbalance sensor. The observed size‐exclusion effect for guest sorption by LLL was found to be broken by the same guests that can change the initial crystal shape. This helps to explain the morphology changes of LLL crystals after the guest sorption and release. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

东海剧毒卡尔藻的形态特征及其系统进化分析

利用光学显微镜、荧光显微镜、扫描电镜及分子生物学等方法, 对分布于我国东海海域的剧毒卡尔藻（Karlodinium veneficum）藻株（LAMB090611）的形态特征和显微结构进行了描述, 并探讨了其分子系统进化关系。该藻株细胞长11.1-18.7 μm, 平均值为（14.2±1.8） μm, 宽8.2-14.7 μm, 平均值为（10.8±1.5） μm。细胞形态结构特征为： 上下锥体积基本相同; 顶沟短而直; 腹孔明显; 纵沟延伸至上锥; 横沟错位距离约占细胞总长的28%-38%; 含有2或4个不规则形态的叶绿体; 细胞核位于中部或下锥。此藻种的暴发可引发有害赤潮（harmful algal bloom）。当前加强有害赤潮的预防和监测工作是减少危害的有效途径, 而对引发赤潮原因种的准确识别和鉴定则是基础和关键。