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.     

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.     

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.     

CRYPTIC AND PSEUDO‐CRYPTIC DIVERSITY IN DIATOMS—WITH DESCRIPTIONS OF PSEUDO‐NITZSCHIA HASLEANA SP. NOV. AND P. FRYXELLIANA SP. NOV.1

A high degree of pseudo‐cryptic diversity was reported in the well‐studied diatom genus Pseudo‐nitzschia. Studies off the coast of Washington State revealed the presence of hitherto undescribed diversity of Pseudo‐nitzschia. Forty‐one clonal strains, representing six different taxa of the P. pseudodelicatissima complex, were studied morphologically using LM and EM, and genetically using genes from three different cellular compartments: the nucleus (D1–D3 of the LSU of rDNA and internal transcribed spacers [ITSs] of rDNA), the mitochondria (cytochrome c oxidase 1), and the plastids (LSU of RUBISCO). Strains in culture at the same time were used in mating studies to study reproductive isolation of species, and selected strains were examined for the production of the neurotoxin domoic acid (DA). Two new species, P. hasleana sp. nov. and P. fryxelliana sp. nov., are described based on morphological and molecular data. In all phylogenetic analyses, P. hasleana appeared as sister taxa to a clade comprising P. calliantha and P. mannii, whereas the position of P. fryxelliana was more uncertain. In the phylogenies of ITS, P. fryxelliana appeared to be most closely related to P. cf. turgidula. Morphologically, P. hasleana differed from most other species of the complex because of a lower density of fibulae, whereas P. fryxelliana had fewer sectors in the poroids and a higher poroid density than most of the other species. P. hasleana did not produce detectable levels of DA; P. fryxelliana was unfortunately not tested. In P. cuspidata, production of DA in offspring cultures varied from higher than the parent cultures to undetectable.     

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.     

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.     

Lichen acclimation to changing environments: Photobiont switching vs. climate‐specific uniqueness in Psora decipiens

Unraveling the complex relationship between lichen fungal and algal partners has been crucial in understanding lichen dispersal capacity, evolutionary processes, and responses in the face of environmental change. However, lichen symbiosis remains enigmatic, including the ability of a single fungal partner to associate with various algal partners. Psora decipiens is a characteristic lichen of biological soil crusts (BSCs), across semi‐arid, temperate, and alpine biomes, which are particularly susceptible to habitat loss and climate change. The high levels of morphological variation found across the range of Psora decipiens may contribute to its ability to withstand environmental change. To investigate Psora decipiens acclimation potential, individuals were transplanted between four climatically distinct sites across a European latitudinal gradient for 2 years. The effect of treatment was investigated through a morphological examination using light and SEM microscopy; 26S rDNA and rbcL gene analysis assessed site‐specific relationships and lichen acclimation through photobiont switching. Initial analysis revealed that many samples had lost their algal layers. Although new growth was often determined, the algae were frequently found to have died without evidence of a new photobiont being incorporated into the thallus. Mycobiont analysis investigated diversity and determined that new growth was a part of the transplant, thus, revealing that four distinct fungal clades, closely linked to site, exist. Additionally, P. decipiens was found to associate with the green algal genus Myrmecia, with only two genetically distinct clades between the four sites. Our investigation has suggested that P. decipiens cannot acclimate to the substantial climatic variability across its environmental range. Additionally, the different geographical areas are home to genetically distinct and unique populations. The variation found within the genotypic and morpho‐physiological traits of P. decipiens appears to have a climatic determinant, but this is not always reflected by the algal partner. Although photobiont switching occurs on an evolutionary scale, there is little evidence to suggest an active environmentally induced response. These results suggest that this species, and therefore, other lichen species, and BSC ecosystems themselves may be significantly vulnerable to climate change and habitat loss.     

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.     

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.     

Identification and Molecular Characterizations of Neoscytalidium dimidiatum Causing Stem Canker of Red‐fleshed Dragon Fruit (Hylocereus polyrhizus) in Malaysia

During the year 2008 to 2009, a new disease of stem canker was noticed in most red‐fleshed dragon fruit (Hylocereus polyrhizus) plantations in Malaysia. The symptoms observed were small circular sunken orange spot, black pycnidia and rotted stem. This study was conducted to determine the occurrence of the stem canker on H. polyrhizus in Malaysia, subsequently to isolate, identify and characterize the fungal pathogen based on morphology and molecular characteristics and pathogenicity test. From the surveyed 20 plantations in Malaysia, stem canker was detected in all the plantations. A total of 40 isolates of Scytalidium‐like fungus were isolated and identified as Neoscytalidium dimidiatum based on morphological characteristics and ITS region sequences, which showed 99% similarity to N. dimidiatum (FJ648577). From the phylogenetic analysis using maximum‐likelihood tree, isolates of N. dimidiatum from stem canker of H. polyrhizus were grouped together and did not show any sequence variation. From pathogenicity test, all 40 isolates of N. dimidiatum were pathogenic causing stem canker on H. polyrhizus. To our knowledge, this is the first report of stem canker of H. polyrhizus caused by N. dimidiatum in Malaysia.     

Chemotaxonomic Considerations of the n‐Alkane Composition in Pinus heldreichii,P. nigra,and P. peuce

The n‐alkane composition in the leaf cuticular waxes of natural populations of Bosnian pine (Pinus heldreichii), Austrian pine (P. nigra), and Macedonian pine (P. peuce) was compared for the first time. The range of n‐alkanes was wider in P. nigra (C₁₆ – C₃₃) than in P. heldreichii and P. peuce (C₁₈ – C₃₃). Species also diverged in abundance and range of dominant n‐alkanes (P. heldreichii: C₂₃, C₂₇, and C₂₅; P. nigra: C₂₅, C₂₇, C₂₉, and C₂₃; P. peuce: C₂₉, C₂₅, C₂₇, and C₂₃). Multivariate statistical analyses (PCA, DA, and CA) generally pointed out separation of populations of P. nigra from populations of P. heldreichii and P. peuce (which were, to a greater or lesser extent, separated too). However, position of these species on the basis of n‐alkane composition was in accordance neither with infrageneric classification nor with recent molecular and terpene investigations.