Miguel de Salas

Takayama Gen, Nov (Gymnodiniales, Dinophyceae), a new genus of unarmored Dinoflagellates with sigmoid apical grooves, including the description of two new species

A new potentially ichthyotoxic dinoflagellate genus, Takayama de Salas, Bolch, Botes et Hallegraeff gen. nov., is described with two new species isolated from Tasmanian (Australia) and South African coastal waters: T. tasmanica de Salas, Bolch et Hallegraeff, sp. nov. and T. helix, de Salas, Bolch, Botes et Hallegraeff, sp. nov. The genus and two species are characterized by LM and EM of field samples and laboratory cultures as well as large subunit rDNA sequences and HPLC pigment analyses of several cultured strains. The new Takayama species have sigmoid apical grooves and contain fucoxanthin and its derivatives as the main accessory pigments. Takayama tasmanica is similar to the previously described species Gymnodinium pulchellum Larsen, Gyrodinium acrotrochum Larsen, and G. cladochroma Larsen in its external morphology but differs from these in having two ventral pores, a large horseshoe‐shaped nucleus, and a central pyrenoid with radiating chloroplasts that pass through the nucleus. It contains gyroxanthin‐diester and a gyroxanthin‐like accessory pigment, both of which are missing in T. helix. Takayama helix has an apical groove that is nearly straight while still being clearly inflected. A ventral pore or slit is present. It has numerous peripheral, strap shaped, and spiraling chloroplasts with individual pyrenoids and a solid ellipsoidal nucleus. The genus Takayama has close affinities to the genera Karenia and Karlodinium.

Lipid, fatty acid, and sterol composition of eight species of Kareniaceae (dinophyta): chemotaxonomy and putative lipid phycotoxins

The lipid class, fatty acid, and sterol composition of eight species of ichthyotoxic marine gymnodinioid dinoflagellate (Karenia, Karlodinium, and Takayama) species was examined. The major lipid class in all species was phospholipid (78%–95%), with low levels of triacylglycerol (TAG; 0%–16%) and free fatty acid (FFA; 1%–11%). The common dinoflagellate polyunsaturated fatty acids (PUFA), octadecapentaenoic acid (OPA 18:5ω3), and docosahexaenoic acid (DHA 22:6ω3), were present in all species in varying amounts (14%–35% and 8%–23%, respectively). The very‐long‐chain PUFA (VLC‐PUFA) 28:7ω6 and 28:8ω3 were present at low levels (<1%), and the ratio of these fatty acids may be a useful chemotaxonomic marker at the species level. The typical dinoflagellate sterol dinosterol was absent from all species tested. A predominance of the 4‐methyl and 4‐desmethyl Δ8(14) sterols in all dinoflagellate species included 23‐methyl‐27‐norergosta‐8(14),22‐dien‐3β‐ol (Karenia papilionacea A. J. Haywood et Steid, 59%–66%); 27‐nor‐(24R)‐4α‐methyl‐5α‐ergosta‐8(14),22‐dien‐3β‐ol, brevesterol, (Takayama tasmanica de Salas, Bolch et Hallegraeff 84%, Takayama helix de Salas, Bolch, Botes et Hallegraeff 71%, Karenia brevis (C. C. Davis) G. Hansen et Moestrup 45%, Karlodinium KDSB01 40%, Karenia mikimotoi (Miyake et Kominami ex Oda) G. Hansen et Moestrup 38%); and (24R)‐4α‐methyl‐5α‐ergosta‐8(14),22‐dien‐3β‐ol, gymnodinosterol, (K. mikimotoi 48%, Karenia umbella de Salas, Bolch et Hallegraeff 59%, Karlodinium veneficum (D. L. Ballant.) J. Larsen 71%–83%). In Takayama species, five steroid ketones were identified, including for the first time the 3‐keto form of brevesterol and gymnodinosterol. These results indicate a biochemical link between sterol and steroid ketone biosynthesis, suggesting that selected dinoflagellates can make a significant contribution to ketones in marine sediments. The presence of steroid ketones, specific sterols, and fatty acids, and the ratio of VLC‐PUFA may prove to be a useful chemotaxonomic tool for distinguishing between morphologically similar species. The relative levels of the PUFA, OPA, and DHA, coupled with the potential inhibitory action of Δ8(14) sterols, may provide an insight into the ichthyotoxicity of these bloom‐forming dinoflagellates.

Description of a New Planktonic Mixotrophic Dinoflagellate Paragymnodinium shiwhaense n. gen., n. sp. from the Coastal Waters off Western Korea: Morphology, Pigments, and Ribosomal DNA Gene Sequence

ABSTRACT. The mixotrophic dinoflagellate Paragymnodinium shiwhaense n. gen., n. sp. is described from living cells and from cells prepared by light, scanning electron, and transmission electron microscopy. In addition, sequences of the small subunit (SSU) and large subunit (LSU) rDNA and photosynthetic pigments are reported. The episome is conical, while the hyposome is hemispherical. Cells are covered with polygonal amphiesmal vesicles arranged in 16 rows and containing a very thin plate‐like component. There is neither an apical groove nor apical line of narrow plates. Instead, there is a sulcal extension‐like furrow. The cingulum is as wide as 0.2–0.3 × cell length and displaced by 0.2–0.3 × cell length. Cell length and width of live cells fed Amphidinium carterae were 8.4–19.3 and 6.1–16.0 μm, respectively. Paragymnodinium shiwhaense does not have a nuclear envelope chamber nor a nuclear fibrous connective (NFC). Cells contain chloroplasts, nematocysts, trichocysts, and peduncle, though eyespots, pyrenoids, and pusules are absent. The main accessory pigment is peridinin. The sequence of the SSU rDNA of this dinoflagellate (GenBank AM408889) is 4% different from that of Gymnodinium aureolum, Lepidodinium viride, and Gymnodinium catenatum, the three closest species, while the LSU rDNA was 17–18% different from that of G. catenatum, Lepidodinium chlorophorum, and Gymnodinium nolleri. The phylogenetic trees show that this dinoflagellate belongs within the Gymnodinium sensu stricto clade. However, in contrast to Gymnodinium spp., cells lack nuclear envelope chambers, NFC, and an apical groove. Unlike Polykrikos spp., which have a taeniocyst–nematocyst complex, P. shiwhaense has nematocysts without taeniocysts. In addition, P. shiwhaense does not have ocelloids in contrast to Warnowia spp. and Nematodinium spp. Therefore, based on morphological and molecular analyses, we suggest that this taxon is a new species, also within a new genus.

Ultrastructure and large subunit rDNA sequences of Lepidodinium viride reveal a close relationship to Lepidodinium chlorophorum comb. nov. (= Gymnodinium chlorophorum )

The ultrastructure of the green dinoflagellate Lepididodinium viride M. M. Watanabe, S. Suda, I. Inouye Sawaguchi et Chihara was studied in detail. The nuclear envelope possessed numerous chambers each furnished with a nuclear pore, a similar arrangement to that found in other gymnodinioids. The flagellar apparatus was essentially identical to Gymnodinium chlorophorum Elbrächter et Schnepf, a species also containing chloroplasts of chlorophyte origin. Of particular interest was the connection of the flagellar apparatus to the nuclear envelope by means of both a fiber and a microtubular extension of the R3 flagellar root. This feature has not been found in other dinoflagellates and suggests a close relationship between these two species. This was confirmed by phylogenetic analysis based on partial sequences of the large subunit (LSU) rDNA gene of L. viride, G. chlorophorum and 16 other unarmoured dinoflagellates, including both the ‘type’ culture and a new Tasmanian isolate of G. chlorophorum. These two isolates had identical sequences and differed from L. viride by only 3.75% of their partial LSU sequences, considerably less than the difference between other Gymnodinium species. Therefore, based on ultrastructure, pigments and partial LSU rDNA sequences, the genus Lepidodinium was emended to encompass L. chlorophorum comb. nov.

Development of a Real-Time PCR Probe for Quantification of the Heterotrophic Dinoflagellate Cryptoperidiniopsis brodyi (Dinophyceae) in Environmental Samples

ABSTRACT A TaqMan format real-time PCR probe was developed against the internal transcribed spacer 2 ribosomal DNA region for the specific detection and quantification of Cryptoperidiniopsis brodyi in environmental samples. The assay specificity was confirmed by testing against related dinoflagellates and verified by sequencing PCR amplicons from natural water samples. Phylogenetic analysis of the sequenced environmental samples also showed that this assay is specific to C. brodyi. The C. brodyi-specific assay was used in conjunction with Pfiesteria piscicida- and Pfiesteria shumwayae-specific real-time PCR assays to investigate the temporal variations of C. brodyi, P. piscicida, and P. shumwayae abundance in the Derwent estuary, Tasmania. The 18-month field survey from November 2004 to April 2006 revealed that C. brodyi occurred in all seasons at very low densities, mostly below 25 cells liter−1, with higher abundance (maximum, 112 cells liter−1) in April and May. P. piscicida was detected only once, in May 2005 at 60 cells liter−1. P. shumwayae was not detected during the survey.

NOVEL UNARMORED DINOFLAGELLATES FROM THE TOXIGENIC FAMILY KARENIACEAE (GYMNODINIALES): FIVE NEW SPECIES OF KARLODINIUM AND ONE NEW TAKAYAMA FROM THE AUSTRALIAN SECTOR OF THE SOUTHERN OCEAN1

Six new species of unarmored dinoflagellates in the family Kareniaceae were isolated from the Australian sector of the Southern Ocean in March 2006: Takayama tuberculata de Salas sp. nov, Karlodinium antarcticum de Salas sp. nov., Karl. ballantinum de Salas sp. nov., Karl. conicum de Salas sp. nov., Karl. corrugatum de Salas sp. nov., and Karl. decipiens de Salas et Laza‐Martínez sp. nov. These new taxa were characterized using light and electron microscopy and sequencing of the LSU rDNA and are well supported based either on their morphology or molecular phylogeny. Takayama tuberculata, isolated just north of the polar front (55°–57° S), is genetically close to T. tasmanica, but smaller, with a significantly reduced number of amphiesmal vesicles. Medium‐sized Karl. antarcticum, also isolated from near the polar front, is characterized by its long ovoid cell outline and very long apical groove. The small Karl. ballantinum has a very short apical groove. The large Karl. conicum has a distinct conical epicone and spherical posterior nucleus. The small Karl. corrugatum, from just south of the polar front, has distinctive parallel striations on the epicone surface and a distinctively shaped and placed ventral pore. The large and widespread Karl. decipiens, distributed through Southern Ocean waters from the polar front to Tasmanian coastal waters, and coastal Spain, has a helicoidal chloroplast arrangement and a large central nucleus. This study represents the first description of species in the potentially ichthyotoxic family Kareniaceae recorded from the Southern Ocean.

Karlodinium australe sp. nov. (Gymnodiniales, Dinophyceae), a new potentially ichthyotoxic unarmoured dinoflagellate from lagoonal habitats of south-eastern Australia

M.F. de Salas, C.J.S. Bolch and G.M. Hallegraeff. 2005. Karlodinium australe sp. nov. (Gymnodiniales, Dinophyceae), a new potentially ichthyotoxic unarmoured dinoflagellate from lagoonal habitats of south-eastern Australia. Phycologia 44: 640–650. A new species in the toxigenic genus Karlodinium, K. australe de Salas, Bolch & Hallegraeff sp. nov., is described from coastal lagoons and enclosed bay habitats in Tasmania and south-eastern Australia. This mixotrophic species is characterised with the use of light and electron microscopy, pigment analysis and sequencing of the large subunit ribosomal gene from field samples and laboratory cultures. Karlodinium australe has a straight, short apical groove and a ventral pore typical of the genus, but is almost twice the size (19–26 µm long) of other species in the genus. It is morphologically similar to Gyrodinium corsicum and Karenia digitata but has an anteriorly located nucleus and a different amphiesmal pattern. Karlodinium australe has a partial large subunit ribosomal gene (26S ribosomal DNA) sequence that differs by 7.2% from that of K. micrum, and its pigment profile does not include gyroxanthindiester. Unlike the type species of the genus, K. micrum, K. australe has no detectable hexagonal array of plug-like structures below the amphiesma, and its chloroplasts contain pyramidal rather than lenticular pyrenoids.

Karenia umbella sp. nov. (Gymnodiniales, Dinophyceae), a new potentially ichthyotoxic dinoflagellate species from Tasmania, Australia

Abstract A new, potentially ichthyotoxic gymnodinioid dinoflagellate, Karenia umbella de Salas, Bolch & Hallegraeff, is described from Tasmanian coastal waters, Australia. It is characterized by light and electron microscopy of field samples and laboratory cultures, as well as large-subunit ribosomal DNA sequences and high-performance liquid chromatography pigment analyses of two cultured strains. The new Karenia species has a straight apical groove and contains fucoxanthin derivative carotenoid pigments, typical of the genus. Karenia umbella differs from its closest relative K. longicanalis in its larger size, longer epicone with a finger-like sulcal intrusion, asymmetrically shaped hypocone, irregularly shaped chloroplasts, and presence of six or eight radial furrows on the epicone surface. It differs from K. digitata in its significantly larger size and the shape of the epicone and hypocone. Genetically, this species is distinct from the dorsoventrally flattened Karenia species K. mikimotoi, K. brevis, and K. brevisulcata.

Karenia asterichroma sp. nov. (Gymnodiniales, Dinophyceae), a new dinoflagellate species associated with finfish aquaculture mortalities in Tasmania, Australia

Abstract A new species of unarmoured dinoflagellate is described from Tasmanian field samples and laboratory cultures: Karenia asterichroma de Salas, Bolch & Hallegraeff, sp. nov. This species is characterized by the use of light and scanning electron microscopy and sequencing of the 26S ribosomal RNA gene. Karenia asterichroma is a dorsoventrally flattened cell resembling K. brevis, although it differs from this species by 4.5% in the D1–D3 regions of its 26S ribosomal DNA (rDNA) sequence. Unlike other Karenia species, the epicone of K. asterichroma has concave sides and the chloroplasts radiate from a central, putative pyrenoid. The nucleus is located in the centre of the epicone and the apical groove is long, extending ventrally to near a broad sulcal extension into the epicone. Partial 26S rDNA sequences indicate a relationship with K. bicuneiformis. Although the ichthyotoxicity of K. asterichroma is yet to be confirmed, it was associated (with three other Karenia species) with a recent mass mortality of over 100,000 caged salmonids at an aquaculture operation in southern Tasmania, Australia.

SURVEY FOR KARLOTOXIN PRODUCTION IN 15 SPECIES OF GYMNODINIOID DINOFLAGELLATES (KARENIACEAE, DINOPHYTA)1

Toxin analysis of 15 species of Kareniaceae revealed the presence of karlotoxin, KmTx 2, in only a single species (Karlodinium veneficum) but with variable activity in strains from the Swan (KmSwanTx 2‐1, 2.1 pg · cell−1; and KmSwanTx 2‐2, 0.53 pg · cell−1), Huon (KmHuonTx 2, 0.86 pg · cell−1), and Derwent rivers (<0.001 pg · cell−1) in Australia. A newly isolated Southern Ocean species, Karlodinium conicum, contained a novel poorly hemolytic karlotoxin analogue (KmconicumTx, 2.8 pg · cell−1). The hemolytic potency (HD50%) of the Australian karlotoxins were as follows: KmSwanTx 2‐1 (65.9 ± 4.8 ng) and KmSwanTx 2‐2 (63.4 ± 3.7 ng), KmHuonTx 2 (343 ± 4.9 ng), and KmconicumTx (>4,000 ng). Species from the closely related genera Takayama (T. helix, T. tasmanica, T. tuberculata), Karenia (K. asterichroma, K. brevis, K. mikimotoi, K. papilionacea, K. umbella), and Karlodinium (Ka. australe, Ka. antarcticum, Ka. ballantinum, Ka. corrugatum, Ka. decipiens) were all consistently negative for karlotoxin production. Brevetoxin (PbTx) was only detected in K. brevis, and hemolytic activity was only observed in Ka. veneficum strains.