Rafael Salas

Amphidoma languida sp. nov. (Dinophyceae) reveals a close relationship between Amphidoma and Azadinium

Toxic algae such as Alexandrium and Azadinium have an important ecological impact and have originated several times independently within the dinophytes. Their closest relatives, however, are mostly unknown at present. A new dinophyte species, Amphidoma languida sp. nov., was isolated from Bantry Bay (Ireland) during a period of elevated azaspiracid toxicity in mussels. The new species was described in detail, and its phylogenetic position was analysed, by using a combination of light and electron microscopy, chemical detection methods, and sequence comparison of concatenated ribosomal RNA sequence data. Morphological similarities, such as cingular and hypothecal plates, the number and arrangement of sulcal plates, and the characteristic apical pore complex with a small X-plate centrally invading the first apical plate, indicated a close relationship between Amphidoma and Azadinium. However, no known azaspiracid analogues were detected in A. languida by liquid chromatography coupled with tandem mass-spectrometry. In a molecular phylogeny, the Amphidomataceae including Amphidoma and Azadinium were an independent lineage among other monophyletic major groups of the dinophytes such as the Suessiales, Prorocentrales, Gonyaulacales, and Peridiniales. Thus, the taxonomic affiliation of Azadinium is clarified, and our data may prove helpful in the development of specific and reliable molecular detection methods of toxic Azadinium.

TAXONOMIC REVISION OF THE DINOFLAGELLATE AMPHIDOMA CAUDATA: TRANSFER TO THE GENUS AZADINIUM (DINOPHYCEAE) AND PROPOSAL OF TWO VARIETIES, BASED ON MORPHOLOGICAL AND MOLECULAR PHYLOGENETIC ANALYSES

The systematic position of Amphidoma caudata Halldal within the genus Amphidoma has remained uncertain as a result of its plate formula and the absence of molecular phylogenetic data. Also, this thecate dinoflagellate taxon has been used to designate two distinct morphotypes. The present study aims to clarify the generic affiliation of Amphidoma caudata and the taxonomic value of two different morphotypes M1 and M2. The new examination of the plate formula using SEM showed that it was the same for both morphotypes and that it corresponded to the tabulation of the recent erected genus Azadinium Elbrächter et Tillmann. Morphometric analysis, using cell size, length of apical projection in conjunction with the cell length, and the ratio of horn and spine showed that M1 and M2 formed two distinct groups. These results were supported by a molecular approach, revealing notable differences in the sequences of LSU rDNA and ITS region between these two morphotypes. Phylogenetic analyses inferred either from LSU and combined SSU, ITS region and COI data positioned M1 and M2 in a sister cluster of Azadinium species while Amphidoma languida Tillmann, Salas et Elbrächter, the only species of Amphidoma for which sequence data were available, was situated in a basal position of the Azadinium clade. Thus, we propose the transfer of Amphidoma caudata to the genus Azadinium and, consequently, the rehabilitation of the original tabulation of the genus Amphidoma Stein. To discriminate the two morphotypes, we propose a rank of variety with the following designations: Azadinium caudatum var. caudatum and Azadinium caudatum var. margalefii.

Real-time PCR Detection of Dinophysis Species in Irish Coastal Waters

Diarrhetic shellfish toxin-producing Dinophysis species occur in Irish coastal waters throughout the year. Dinophysis acuta and Dinophysis acuminata are the most commonly occurring species and are responsible for the majority of closures of Irish mussel farms. This study describes the development of a qualitative real-time polymerase chain reaction (PCR) assay for identification of D. acuta and D. acuminata in Irish coastal waters. DNA sequence information for the D1-D2 region of the large ribosomal sub-unit (LSU) was obtained, following single-cell PCR of D. acuta and D. acuminata cells isolated from Irish coastal locations. PCR primers and hybridization probes, specific for the detection of D. acuta, were designed for real-time PCR on the LightCycler™. The LightCycler™ software melt curve analysis programme determined that D. acuta was identified by a melt-peak at 61°C, while D. acuminata cells produced a melt peak at 48°C. The limit of detection of the real-time PCR assay was determined to be one to ten plasmid copies of the LSU D1-D2 target region for both species and one to five D. acuminata cells. Lugols preserved water samples were also tested with the assay. The real-time PCR assay identified Dinophysis species in 100% of samples found to contain Dinophysis species by light microscopy and had a greater than 90% correlation with light microscopy for identification of D. acuta and D. acuminata in the samples. The assay can identify and discriminate D. acuta and D. acuminata at low numbers in Irish waters and has the potential to add value to the Irish phytoplankton monitoring programme.

Harmful algal bloom forecast system for SW Ireland. Part II: Are operational oceanographic models useful in a HAB warning system

This study investigated the application of a three-dimensional physical hydrodynamic model in a harmful algal bloom forecast system for Bantry Bay, southwest Ireland. Modelled oceanographic conditions were studied and used to help understand observed changes in the chemical and biological patterns from the national biotoxins and phytoplankton monitoring program. The study focused on two toxic events in 2013. An upwelling event was predicted by the model prior to the appearance and population increase of potentially toxic diatoms, Pseudo-nitzschia, and associated domoic acid in shellfish. A downwelling episode was provided as a forecast in the model prior to the arrival of a Dinophysis bloom and detection of its associated biotoxins in Bay shellfish. The modelled forecast products developed included expected surface, mid-depth and bottom current pathways at the mouth of the Bay and on the adjacent shelf. The rate and direction of water volume flow at the mouth and mid-bay sections were produced by the model to examine predicted upwelling and downwelling pulses. The model also calculated the evolution of water properties (temperature, salinity and density) with depth along the Bay axis and on the adjacent continental shelf. Direct measurements of water properties at a fixed point, mid-bay, were comparable to model calculations. The operational model for southwest Ireland produces a reliable 3-day physical hydrodynamic forecast of the dominant regional physical processes that result in water exchange events between Bantry Bay and its adjacent shelf. While simulated physical hydrodynamics were provided as a 3-day forecast, the upwelling and downwelling signals from the model, closely linked to toxic HAB episodes, were evident up to 10 days prior to the contamination of shellfish in the Bay.

Morphological and molecular characterization of the small armoured dinoflagellate Heterocapsa minima (Peridiniales, Dinophyceae)

The dinophycean genus Heterocapsa is of considerable interest as it contains a number of bloom-forming and/or harmful species. Fine structure of organic body scales is regarded as the most important morphological feature for species determination but currently is unknown for the species H. minima described by Pomroy 25 years ago. Availability of a culture of H. minima collected in the south-west of Ireland allowed us to provide important information for this species, including cell size, cell organelle location, thecal plate pattern, body scale fine structure and molecular phylogeny. Light microscopy revealed the presence of one reticulate chloroplast, an elongated centrally located nucleus, and the presence of one pyrenoid surrounded by a starch sheath. Scanning electron microscopy (SEM) of the thecal plate pattern indicated that Pomroy erroneously designated the narrow first cingular plate as a sulcal plate. In addition, SEM revealed as yet unreported details of the apical pore complex and uncommon ornamentations of hypothecal plates. Organic body scales of H. minima were about 400 nm in size, roundish, with a small central hole and one central, six peripheral and three radiating spines. They differ from other body scales described within this genus allowing for positive identification of H. minima. Heterocapsa minima shares gross cell morphological features (hyposome smaller than episome, elongated nucleus in the middle of the cell, one pyrenoid located in the episome on its left side) with H. arctica (both subspecies H. arctica subsp. arctica and H. arctica subsp. frigida), H. lanceolata and H. rotundata. These relationships are reflected in the phylogenetic trees based on LSU and ITS rDNA sequence data, which identified H. arctica (both subspecies), H. rotundata and H. lanceolata as close relatives of H. minima.

Molecular analyses of protists in long-term observation programmes—current status and future perspectives

Protists (microbial eukaryotes) are diverse, major components of marine ecosystems, and are fundamental to ecosystem services. In the last 10 years, molecular studies have highlighted substantial novel diversity in marine systems including sequences with no taxonomic context. At the same time, many known protists remain without a DNA identity. Since the majority of pelagic protists are too small to identify by light microscopy, most are neither comprehensively or regularly taken into account, particularly in Long-term Ecological Research Sites. This potentially undermines the quality of research and the accuracy of predictions about biological species shifts in a changing environment. The ICES Working Group for Phytoplankton and Microbial Ecology conducted a questionnaire survey in 2013–2014 on methods and identification of protists using molecular methods plus a literature review of protist molecular diversity studies. The results revealed an increased use of high-throughput sequencing methods and a recognition that sequence data enhance the overall datasets on protist species composition. However, we found only a few long-term molecular studies and noticed a lack of integration between microscopic and molecular methods. Here, we discuss and put forward recommendations to improve and make molecular methods more accessible to Long-term Ecological Research Site investigators.