Jane Lewis

Sequence comparisons of toxic and non-toxic Alexandrium tamarense (Dinophyceae) isolates from UK waters

Abstract Polymerase chain reaction was used to amplify and sequence the LSU rDNA gene of 21 isolates of Alexandrium tamarense collected from the United Kingdom. Analysis of a 621 bp sequence covering the D1/D2 region of LSU rDNA revealed two distinct genetic lineages. In all cases, the strains shown to be nontoxic by ELISA or high-performance liquid chromatography were assigned to a ‘Western European’ lineage, whereas those producing the paralytic shellfish poisoning (PSP) toxin were assigned to the ‘North American’ lineage, which also included toxic isolates from the Orkneys. Our data are consistent with previous findings that an isolates ability to produce PSP toxin, or lack thereof, correlates with its phylogenetic position and supports the hypothesis that at least two distinct populations of Alexandrium exist in UK waters.

Morphological and LSU rDNA sequence variation within the Gonyaulax spinifera-Spiniferites group (Dinophyceae) and proposal of G-elongata comb. nov and G-membranacea comb. nov

Abstract Cultures were established from cysts of the cyst-based taxa Spiniferites elongatus and S. membranaceus. Motile cells and cysts from both cultures and sediment samples were examined using light and scanning electron microscopy. The cyst–theca relationship was established for S. elongatus. The motile cells have the tabulation pattern 2 pr, 4′, 6″, 6c, ≥ 4s, 6″′, 1p, 1″″, but they remain unattributable to previously described Gonyaulax species. There was large variation in process length and process morphology in cysts from both cultures and wild samples and there was variation in ornamentation and in the development of spines and flanges in motile cells. A new combination, G. elongata (Reid) Ellegaard et al. comb. nov. is proposed, following new rules of the International Code of Botanical Nomenclature that give genera based on extant forms priority over genera based on fossil forms. Extreme morphological variation in the cyst and motile stages of S. membranaceus is described and this species is also transferred to the genus Gonyaulax, as G. membranacea (Rossignol) Ellegaard et al. comb. nov. Approximately 1500 bp of large subunit (LSU) rDNA were determined for these two species and for G. baltica, G. cf. spinifera (= S. ramosus) and G. digitalis (= Bitectatodinium tepikiense). LSU rDNA showed sequence divergences similar to those estimated between species in other genera within the Gonyaulacales; a phylogeny for the Gonyaulacales was established, including novel LSU rONA sequences for Alexandrium margalefii, A. pseudogonyaulax and Pyrodinium bahamense var. compressum. Our results show that motile stages obtained from the germination of several cysts of the ‘fossil-based’ Spiniferites and B. tepikiense, which were previously attributed to ‘Gonyaulax spinifera group undifferentiated’, belong to distinct species of the genus Gonyaulax. These species show small morphological differences in the motile stage but relatively high sequence divergence. Moreover, this group of species is monophyletic, supported by bootstrap values of 100% in parsimony and maximum likelihood analyses.

Cyst-theca relationship, life cycle, and effects of temperature and salinity on the cyst morphology of Gonyaulax baltica sp. Nov. (Dinophyceae) from the Baltic Sea area

A new species of Gonyaulax, here named Gonyaulax baltica sp. nov., has been isolated from sediment samples from the southeastern Baltic. Culture strains were established from individually isolated cysts, and cyst formation was induced in a nitrogen‐depleted medium. Although G. baltica cysts are similar to some forms attributed to Spiniferites bulloideus and the motile stage of G. baltica has affinities with G. spinifera, the combination of features of cyst and motile stage of G. baltica is unique. The culture strains were able to grow at salinity levels from 5 to 55 psu and formed cysts from 10 to 50 psu. Cultures at each salinity level were grown at 12, 16, and 20° C. Temperature‐ and salinity‐controlled morphological variability was found in the resting cysts. Central body size varied with temperature and salinity, and process length varied with salinity. Cysts that formed at extreme salinity levels displayed lower average process length than cysts formed at intermediate salinity levels, and central body length and width were lowest at higher temperature and lower salinity. Models for the relationship between central body size and temperature/salinity and process length and salinity have been developed and may be used to determine relative paleosalinity and paleotemperature levels. Our results on salinity‐dependent process length confirm earlier reports on short‐spined cysts of this species found in low salinity environments, and the model makes it possible to attempt to quantify past salinity levels.

THE CYST OF THE CALCAREOUS DINOFLAGELLATE SCRIPPSIELLA TRIFIDA: RESOLVING THE FOSSIL RECORD OF ITS ORGANIC WALL WITH THAT OF ALEXANDRIUM TAMARENSE

Abstract Scrippsiella trifida Lewis, 1991 ex Head, 1996 is a nontoxic marine calciodinelloidean dinoflagellate whose resting cyst has a distinctive wall containing large, erect, trifurcate, recurving calcareous processes that separate two organic layers. We show that the organic wall layers of living Scrippsiella trifida cysts are resistant to acetolysis and can therefore potentially fossilize, and we report on abundant Scrippsiella trifida cysts from latest Pleistocene and early Holocene marine sediments off eastern Canada, representing the first confirmed fossil discovery of this species in the North Atlantic. A reappraisal of late Quaternary palynological records now shows that the organic remains of Scrippsiella trifida cysts have been widely misidentified as cysts of Alexandrium tamarense (Lebour, 1925) Balech, 1985, a goniodomacean (and hence noncalcareous) dinoflagellate and major cause of paralytic shellfish poisoning in humans. The morphology of these two cyst types is contrasted, and the modern and fossil distribution of Scrippsiella trifida cysts in sediments of the North Atlantic and adjacent areas is now clarified. It is apparent from this distribution that Scrippsiella trifida favors neritic environments characterized by cool winters and relatively warm (14°–25°C) summers. Extremely high fluxes of S. trifida cysts in nearshore areas off Nova Scotia and southern Greenland during deglaciation and early postglacial time (14–7 ka) have no modern analog but may signal a reduction in salinity caused by meltwater discharge. In general, the organic walls of calcareous dinoflagellate cysts are more common components of palynological assemblages than hitherto realized.

Preliminary observations of cyst-theca relationships in Spiniferites ramosus and Spiniferites membranaceus (Dinophyceae)

Motile thecal cells derived from the hatching of single cysts identified as Spiniferites membranaceus and S. ramosus have been used to establish cultures. These cultures were examined in order to assess the cyst-theca relationships of these two taxa. The cultures produced two different motile Gonyaulax species belonging to Kofoids Spinifera group. These cultures were then induced to form a new cyst generation under uniform conditions, and examination of large numbers of the resulting cysts has shown that process development is an extremely variable phenomenon although process morphologies display a continuum within a species. Process length (and to a certain degree, process morphology) requires careful interpretation when used to discriminate Spiniferites taxa, in both modern and ancient environments.

Thalassiosira species (Bacillariophyceae) from a Scottish sea-loch

During the spring diatom blooms of 1992 and 1993 in Loch Creran, Scotland, regular water column and sediment samples were collected, examined, and species composition of the phytoplankton determined. Thalassiosira species were found to be a major bloom constituent. Species were identified from specimens cultured from sediments and isolated from vertical net hauls, using light and scanning electron microscopy. Of the 17 species positively determined, only Thalassiosira angulata, T. anguste-lineata, T. eccentrica, T. gravida, T. minima, T. nordenskioeldii, T. rotula and T. tenera have been previously reported in Scottish waters. Other species, for example Thalassiosira pseudonana and T. punctigera, have been noted elsewhere in the British Isles but not along the Scottish west coast. Several species, namely Thalassiosira concaviuscula, T. constricta, T. delicatula, T. kuschirensis, T. oceanica, T. pacifica and T. tealata, are new records for the British Isles. One unidentified taxon is illustrated. Descriptions, illustrations and a key are provided for the 18 taxa.

In situ identification and localization of bacteria associated with Gyrodinium instriatum (Gymnodiniales, Dinophyceae) by electron and confocal microscopy

The presence of intracellular bacteria in the dinoflagellate Gyrodinium instriatum Freudenthal & Lee has previously been described but the bacterial flora associated with this species has not been characterized. In this study, new results of transmission electron microscopy (TEM) and in situ hybridization using several bacterial group-specific oligonucleotide probes are presented. The long-term association of endocytoplasmic and endonuclear bacteria with G. instriatum has been confirmed. All endonuclear and most of the endocytoplasmic bacteria labelled were identified as belonging to the betaproteobacteria. Large clusters of Cytophaga-Flavobacterium-Bacteroides (CFB) were labelled and observed in the cytoplasm of the dinoflagellate cells, but were absent from the nucleus. Gammaproteobacteria were only observed outside the dinoflagellates. No alphaproteobacteria were detected either free-living or intracellular. Empirical observation of intracellular CFB reflected a degradation process of moribund dinoflagellate cells, whereas the systematic colonization of dinoflagellate nucleoplasm by betaproteobacteria suggested a true symbiotic relationship. Natural colonization may have occurred, perpetuated by vertical transmission of intracellular bacteria to the dinoflagellate daughter cells, via a pool of bacteria sequestered within the nucleus. Dividing bacteria were observed in the nucleus and equilibrium may be maintained by release of endonuclear bacteria to the cytoplasm through nuclear envelope constrictions.

Bacterium-dinoflagellate interactions: investigative microscopy of Alexandrium spp. (Gonyaulacales, Dinophyceae)

Abstract The association of bacteria with dinoflagellates has been a neglected field of study, which has gained prominence in recent years because of the possible role of bacteria in toxin synthesis. A number of dinoflagellates undergo sexual reproduction, passing through various life-cycle stages in addition to the vegetative form. The presence of bacteria within dinoflagellates has been well established, but their presence throughout the dinoflagellate life-cycle has not been investigated. Using cultures of Alexandrium (A. tamarense, A. fundyense), we investigated the association of bacteria with various vegetative growth phases (lag, log, stationary) and sexual life-cycle stages (planozygote, planomeiocyte, hypnozygote), using scanning electron microscopy, transmission electron microscopy (TEM) and epifluorescence microscopy. Bacteria were found to be associated with the surfaces of vegetative cells, planozygotes, hypnozygotes and planomeiocytes. TEM showed bacteria to be present within all vegetative growth phases, as well as in the sexual planozygote, cyst and planomeiocyte. The presence of intra-cellular bacteria in vegetative cells was also confirmed using DAPI staining combined with epifluorescence microscopy, and lipopolysaccharide staining combined with TEM.

An assessment of the accuracy and reproducibility of the most probable number (MPN) technique in estimating numbers of nutrient stressed diatoms in sediment samples

A series of experiments was carried out to determine the accuracy and reproducibility of the most probable number (MPN) technique for estimating concentrations of viable planktonic diatoms from sediment samples. Two contrasting species were used to inoculate sterilised sediments: Ditylum brightwellii (large single-celled species) and Skeletonema costatum (small chain-forming species). MPN estimates were carried out on sediment samples that had been inoculated with known numbers of cells to produce three different cell concentrations for each species. The results obtained for these samples were predominantly underestimates of cell concentration for both species. The method was insensitive to low cell concentrations (less than or equal to 50 ml(-1)). Results at higher concentrations indicate that the technique is best treated as a semiquantitative method for determining relative abundance.

The palynological expressions of post-Palaeogene upwelling: a review

Abstract This paper reviews the known palynological expressions of post-Palaeogene upwelling, and changes in upwelling. In Plio-Pleistocene upwelling regimes, levels of amorphogen tend to swamp other palynoclasts present; the application of transmission electron microscopy has great potential for elucidating the origins of amorphogen. The recovery of foraminiferal test linings may be controlled by their progressive microbial destruction; the more juvenile stages in the ontogenetic record stand the greatest chance of preservation. The study of dinoflagellate cyst biofacies offers the greatest potential for palynological characterization and interpretation of post-Palaeogene upwelling systems. Cysts of heterotrophic peridiniacean dinoflagellates (P-cysts) tend to dominate cysts of autotrophic gonyaulacacean forms (G-cysts). The ratio of P-cysts to G-cysts (P/G ratio) is therefore considered to be a useful indicator of variable upwelling strength in these settings, reflecting the degree of water column turbulence. It is uncertain to what degree alynological patterns recognizable in one system are representative of other contemporaneous upwelling regimes. Nevertheless, palynological studies of upwelling systems should be used to interpret geochemical data and thereby provide a tangible link between cause (palaeobiological) and effect (geochemical).