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.     

Taxonomical diversity of Pseudo‐nitzschia from the Central Adriatic Sea

The aim of our research was to study the composition of Pseudo‐nitzschia species during a period when neurotoxin domoic acid (DA) was present in shellfish. Sampling was conducted in Ka?tela Bay (Central Adriatic Sea), between November 2015 and January 2016. Concentrations of DA analyzed in various shellfish species were low, below the regulatory limit, while the highest abundance of Pseudo‐nitzschia spp. reached 1.85 × 10⁵ cells L^?1 in the surface layer, at the beginning of November. Within the temperature and salinity range recorded during the investigated period, a positive correlation of Pseudo‐nitzschia spp. abundance was recorded with temperature. Morphological analyses by scanning electron microscopy revealed the presence of five Pseudo‐nitzschia species that had already been reported in the Adriatic Sea – P. calliantha, P. delicatissima, P. fraudulenta, P. pseudodelicatissima /P. cuspidata and P. subfraudulenta – as well as an unknown Pseudo‐nitzschia sp. The composition of the Pseudo‐nitzschia assemblage changed over the investigated period. The species P. pseudodelicatissima/P.cuspidata was found throughout the entire period and the highest diversity was noticed in January, when all six observed species were recorded. These results represent the first taxonomical investigation of the genus Pseudo‐nitzschia in Ka?tela Bay, as well as the first report of DA in shellfish from this area.     

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.     

IDENTIFICATION AND ASSESSMENT OF DOMOIC ACID PRODUCTION IN OCEANIC PSEUDO‐NITZSCHIA (BACILLARIOPHYCEAE) FROM IRON‐LIMITED WATERS IN THE NORTHEAST SUBARCTIC PACIFIC1

We identified and investigated the potential toxicity of oceanic Pseudo‐nitzschia species from Ocean Station Papa (OSP), located in a high‐nitrate, low‐chlorophyll (HNLC) region of the northeast (NE) subarctic Pacific Ocean. Despite their relatively low abundances in the indigenous phytoplankton assemblage, Pseudo‐nitzschia species richness is high. The morphometric characteristics of five oceanic Pseudo‐nitzschia isolates from at least four species are described using SEM and TEM. The species identified are Pseudo‐nitzschia dolorosa Lundholm et Moestrup, P. granii Hasle, P. heimii Manguin, and P. cf. turgidula (Hust.) Hasle. Additional support for the taxonomic classifications based on frustule morphology is provided through the sequencing of the internal transcribed spacer 1 (ITS1) rDNA. Pseudo‐nitzschia species identification was also assessed by the construction of ITS1 clone libraries and using automated ribosomal intergenic spacer analysis (ARISA) for environmental samples collected during the Subarctic Ecosystem Response to Iron Enrichment Study (SERIES), conducted in close proximity to OSP in July of 2002. Based on ITS1 sequences, the presence of P. granii, P. heimii, P. cf. turgidula, and at least five other putative, unidentified Pseudo‐nitzschia ITS1 variants was confirmed within iron‐enriched phytoplankton assemblages at OSP. None of the oceanic isolates produced detectable levels of particulate domoic acid (DA) when in prolonged stationary phase due to silicic acid starvation. The lack of detectable concentrations of DA suggests that either these strains produce very little or no toxin, or that the physiological conditions required to promote particulate DA production were not met and thus differ from their coastal, toxigenic congeners.     

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.     

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.     

Differential Influence of Life Cycle on Growth and Toxin Production of three Pseudo‐nitzschia Species (Bacillariophyceae)

We used a multistrain approach to study the intra‐ and interspecific variability of the growth rates of three Pseudo‐nitzschia species – P. australis, P. fraudulenta, and P. pungens – and of their domoic acid (DA) production. We carried out mating and batch experiments to investigate the respective effects of strain age and cell size, and thus the influence of their life cycle on the physiology of these species. The cell size – life cycle relationship was characteristic of each species. The influence of age and cell size on the intraspecific variability of growth rates suggests that these characteristics should be considered cautiously for the strains used in physiological studies on Pseudo‐nitzschia species. The results from all three species do not support the hypothesis of a decrease in DA production with time since isolation from natural populations. In P. australis, the cellular DA content was rather a function of cell size. More particularly, cells at the gametangia stage of their life cycle contained up to six times more DA than smaller or larger cells incapable of sexual reproduction. These findings reveal a link between P. australis life cycle and cell toxicity. This suggest that life cycle dynamics in Pseudo‐nitzschia natural populations may influence bloom toxicity.     

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.     

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.     

INTER‐ AND INTRASPECIFIC COMMUNITY STRUCTURE WITHIN THE DIATOM GENUS PSEUDO‐NITZSCHIA (BACILLARIOPHYCEAE)1

Pseudo‐nitzschia‐specific PCR primers (PnAll F/R) were designed to amplify a polymorphic region of the internal transcribed spacer 1 (ITS1) from at least 11 Pseudo‐nitzschia species. The primers were used to generate environmental clone libraries from Puget Sound, Washington, and Vancouver Island, British Columbia, to confirm that the primers were specific for Pseudo‐nitzschia and to determine the extent of ITS1 sequence diversity within individual species. All environmental ITS1 sequences generated with PnAll primers displayed the greatest similarity to known Pseudo‐nitzschia ITS1 sequences. The length of cloned ITS1 fragments differed among species but was conserved within a species. Intraspecific genotypes exhibited <3% sequence divergence for seven of the 10 species detected in clone libraries. Several ITS1 genotypes unique to the Pacific Northwest were identified in environmental samples, and other genotypes were more broadly distributed. The Pseudo‐nitzschia primers were also used to develop an automated ribosomal intergenic spacer analysis (ARISA) to rapidly identify Pseudo‐nitzschia species in environmental samples based on species‐specific variation in the length of the targeted ITS1 region. The ARISA peaks were then associated with the environmental clone sequences for Pseudo‐nitzschia species. Surveying the genetic composition of communities at both the inter‐ and intraspecific levels will enhance our understanding of Pseudo‐nitzschia bloom dynamics.     

The diatom genus Pseudo‐nitzschia (Bacillariophyceae) in New South Wales,Australia: morphotaxonomy,molecular phylogeny,toxicity, and distribution

Species belonging to the potentially harmful diatom genus Pseudo‐nitzschia, isolated from 16 localities (31 sampling events) in the coastal waters of south‐eastern Australia, were examined. Clonal isolates were characterized by (i) light and transmission electron microscopy; (ii) phylogenies, based on sequencing of nuclear‐encoded ribosomal deoxyribonucleic acid (rDNA) regions and, (iii) domoic acid (DA) production as measured by liquid chromatography–mass spectrometry (LC‐MS/MS). Ten taxa were unequivocally confirmed as Pseudo‐nitzschia americana, P. arenysensis, P. calliantha, P. cuspidata, P. fraudulenta, P. hasleana, P. micropora, P. multiseries, P. multistriata, and P. pungens. An updated taxonomic key for south‐eastern Australian Pseudo‐nitzschia is presented. The occurrence of two toxigenic species, P. multistriata (maximum concentration 11 pg DA per cell) and P. cuspidata (25.4 pg DA per cell), was documented for the first time in Australia. The Australian strains of P. multiseries, a consistent producer of DA in strains throughout the world, were nontoxic. Data from 5,888 water samples, collected from 31 oyster‐growing estuaries (2,000 km coastline) from 2005 to 2009, revealed 310 regulatory exceedances for “Total Pseudo‐nitzschia,” resulting in six toxic episodes. Further examination of high‐risk estuaries revealed that the “P. seriata group” had highest cell densities in the austral summer, autumn, or spring (species dependent), and lowest cell densities in the austral winter, while the “P. delicatissima group” had highest in winter and spring.     

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.