Genetic diversity of Gambierdiscus spp. (Gonyaulacales, Dinophyceae) in Japanese coastal areas

The genetic diversity of the ciguatera fish poisoning-related dinoflagellate distributed in Japanese coastal areas was investigated. The entire sequence of the 5.8S rRNA gene and two internal transcribed (ITS) regions were determined, which included putative pseudogenes, from 19 strains of dinoflagellates assigned to the genus Gambierdiscus Adachi et Fukuyo collected from Japanese subtropical and temperate coastal areas. The sequences obtained from the 19 strains were divided into two types based on sequence similarity. Here we designate the two types as type 1 and type 2. For the relationship between the genotypes and origins of the strains used, the strains collected from subtropical areas possessed the type 1 sequence; whereas those from temperate areas possessed the type 2. This observation led us to question former reputations that Gambierdiscus cells observed in Japanese temperate areas are immigrants from Japanese subtropical areas. Subsequently, we sequenced a part of the 18S rRNA gene from two strains from subtropical areas and two from temperate areas. Unfortunately, phylogenetic analysis including the sequences obtained from various gonyaulacales dinoflagellates failed to determine the species phylogenetically closely related to and possible origin(s) of the Gambierdiscus sp. from the Japanese coastal areas.     

Pleurosigma hinzianum sp. nov. and P. frenguellianum sp. nov. (Pleurosigmataceae,Bacillariophyta) from Argentinean coastal waters,in comparison with P. amara Stidolph and P. elongatum W. Smith

Two new marine diatom species from Argentinean coastal waters, Pleurosigma hinzianum Sterrenburg, Sunesen & Sar, sp. nov. and P. frenguellianum Sunesen, Sterrenburg & Sar, sp. nov., are described. The characters permitting their identification are specified, based on comparison with type material of the morphologically similar species P. amara Stidolph and P. elongatum W. Smith in the light (LM) and scanning electron microscope (SEM). New information on the type material of P. elongatum is presented and its taxonomic concept is emended. The main criteria for separation of the species discussed here are: valve outline, path of the raphe-sternum, raphe angle and angle of intersection of the oblique striae in LM; and external central and terminal raphe fissures, internal details of the central area, external and internal apical structure, and morphology of the hymen-occluded internal pores in SEM. The occurrence of these (and probably other) species in the genus Pleurosigma in net samples is adventitious and not indicative of a true planktonic mode of life.     

Taxonomic assignment of the benthic toxigenic dinoflagellate Gambierdiscus sp. type 6 as Gambierdiscus balechii (Dinophyceae), including its distribution and ciguatoxicity

Recent molecular phylogenetic studies of Gambierdiscus species flagged several new species and genotypes, thus leading to revitalizing its systematics. The inter-relationships of clades revealed by the primary sequence information of nuclear ribosomal genes (rDNA), however, can sometimes be equivocal, and therefore, in this study, the taxonomic status of a ribotype, Gambierdiscus sp. type 6, was evaluated using specimens collected from the original locality, Marakei Island, Republic of Kiribati; and specimens found in Rawa Island, Peninsular Malaysia, were further used for comparison. Morphologically, the ribotype cells resembled G. scabrosus, G. belizeanus, G. balechii, G. cheloniae and G. lapillus in thecal ornamentation, where the thecal surfaces are reticulate-foveated, but differed from G. scabrosus by its hatchet-shaped Plate 2′, and G. belizeanus by the asymmetrical Plate 3′. To identify the phylogenetic relationship of this ribotype, a large dataset of the large subunit (LSU) and small subunit (SSU) rDNAs were compiled, and performed comprehensive analyses, using Bayesian-inference, maximum-parsimony, and maximum-likelihood, for the latter two incorporating the sequence-structure information of the SSU rDNA. Both the LSU and SSU rDNA phylogenetic trees displayed an identical topology and supported the hypothesis that the relationship between Gambierdiscus sp. type 6 and G. balechii was monophyletic. As a result, the taxonomic status of Gambierdiscus sp. type 6 was revised, and assigned as Gambierdiscus balechii. Toxicity analysis using neuroblastoma N2A assay confirmed that the Central Pacific strains were toxic, ranging from 1.1 to 19.9 fg P-CTX-1 eq cell⁻¹, but no toxicity was detected in a Western Pacific strain. This suggested that the species might be one of the species contributing to the high incidence rate of ciguatera fish poisoning in Marakei Island.     

Nocardia testaceus sp. nov. and Nocardia senatus sp. nov., isolated from patients in Japan

Two actinomycete strains isolated from sputum between 1999 and 2001 in Japan were provisionally assigned to the genus Nocardia based on morphological criteria. These isolates were further studied in order to determine their specific taxonomic status. Detailed chemotaxonomic characterization and 16S rDNA gene sequence analysis of these isolates also confirmed that they belong to the genus Nocardia. The 16S rDNA sequence data of the two strains showed that they are most similar to that of Nocardia carnea and Nocardia flavorosea. However, DNA-DNA relatedness data showed that the two strains could be distinguished from N. carnea and N. flavorosea and therefore represented two new species within the genus Nocardia. The designation of the two isolated strains are Nocardia testaceus for IFM 0937(T) (=JCM 12235(T), DSM 44765(T)) and Nocardia senatus for IFM 10088(T) (=JCM 12236(T), DSM 44766(T)).     

Characterization of Gambierdiscus lapillus sp. nov. (Gonyaulacales,Dinophyceae): a new toxic dinoflagellate from the Great Barrier Reef (Australia)

Gambierdiscus is a genus of benthic dinoflagellates found worldwide. Some species produce neurotoxins (maitotoxins and ciguatoxins) that bioaccumulate and cause ciguatera fish poisoning (CFP), a potentially fatal food‐borne illness that is common worldwide in tropical regions. The investigation of toxigenic species of Gambierdiscus in CFP endemic regions in Australia is necessary as a first step to determine which species of Gambierdiscus are related to CFP cases occurring in this region. In this study, we characterized five strains of Gambierdiscus collected from Heron Island, Australia, a region in which ciguatera is endemic. Clonal cultures were assessed using (i) light microscopy; (ii) scanning electron microscopy; (iii) DNA sequencing based on the nuclear encoded ribosomal 18S and D8‐D10 28S regions; (iv) toxicity via mouse bioassay; and (v) toxin profile as determined by Liquid Chromatography‐Mass Spectrometry. Both the morphological and phylogenetic data indicated that these strains represent a new species of Gambierdiscus, G. lapillus sp. nov. (plate formula Po, 3′, 0a, 7″, 6c, 7‐8s, 5?, 0p, 2″″ and distinctive by size and hatchet‐shaped 2′ plate). Culture extracts were found to be toxic using the mouse bioassay. Using chemical analysis, it was determined that they did not contain maitotoxin (MTX1) or known algal‐derived ciguatoxin analogs (CTX3B, 3C, CTX4A, 4B), but that they contained putative MTX3, and likely other unknown compounds.     

Amphidiniopsis uroensis sp. nov. and Amphidiniopsis pectinaria sp. nov. (Dinophyceae): Two new benthic dinoflage llates from Japan

Two new armoured, heterotrophic sand‐dwelling marine dinoflagellates, Amphidiniopsis uroensis Toriumi, Yoshimatsu et Dodge sp. nov. and Amphidiniopsis pectinaria Toriumi, Yoshimatsu et Dodge sp. nov. were collected from Japanese sandy beaches, and their morphological features observed by light microscopy and scanning electron microscopy (SEM). The cell size of A. uroensis is 28–31 μm in length and 23–28 μm in width. The plate formula is Po 3′, 3a, 6″, 3c, 4s (+1 acc.), 5″′, 2″″. The thecal surface is ornamented with small processes, pores and spines, however, the surface of plate 2a is smooth. The epitheca possesses a narrow ridge that is extended along on the suture between 1′ and 3′. Plate 1″ connects with the right sulcal (Sd) and right sulcal accessory (Sda) plates, so the cingulum is incomplete. A nucleus is situated in the central part of the cell. There are a few small spines at the antapex. There are no stigma or chloroplasts. Amphidiniopsis pectinaria cells are 33–40 urn in length and 29–35 μm in width. The plate formula is Po 4′, 3a, 7″, 3c, 4s (+1 acc.), 5″′, 2″″. Plate 1″ connects directly with Sd and Sda plates, so the cingulum is incomplete. The thecal surface is ornamented with small processes, spines and pores. The epitheca is provided with a narrow ridge that is extended along on the suture between plates 1′, 4′ and 7″. The ornamentation on the antapical plates is unique. It is arranged in 10 straight rows on the hypotheca; each row has a strong spine at its posterior end. In addition, there is a long spine at the antapex. There are no stigma or chloroplasts. A nucleus is located in the central part of the cell.     

MORPHOLOGY AND MOLECULAR ANALYSES OF THREE TOXIC SPECIES OF GAMBIERDISCUS (DINOPHYCEAE): G. PACIFICUS, SP. NOV., G. AUSTRALES, SP. NOV., AND G. POLYNESIENSIS, SP. NOV.

Three new dinoflagellate species, Gambierdiscus polynesiensis, sp. nov., Gambierdiscus australes, sp. nov., and Gambierdiscus pacificus, sp. nov., are described from scanning electron micrographs. The morphology of the three new Gambierdiscus species is compared with the type species Gambierdiscus toxicus Adachi et Fukuyo 1979, and two other species: Gambierdiscus belizeanus Faust 1995 and Gambierdiscus yasumotoi Holmes 1998. The plate formula is: Po, 3′, 7", 6C, 8S, 5‴, 1p, 2". Culture extracts of these three new species displayed both ciguatoxin- and maitotoxin-like toxicities. The following morphological characteristics differentiated each species. 1) Cells of G. polynesiensis are 68–85 μm long and 64–75 μm wide, and the cell’s surface is smooth. They are identified by a large triangular apical pore plate (Po), a narrow fish-hook opening surrounded by 38 round pores, and a large, broad posterior intercalary plate (1p) wedged between narrow postcingular plates 2‴ and 4‴. Plate 1p occupies 60% of the width of the hypotheca. 2) Cells of G. australes also have a smooth surface and are 76–93 μm long and 65–85 μm wide in dorsoventral depth. They are identified by the broad ellipsoid apical pore plate (Po) surrounded by 31 round pores and a long and narrow 1p plate wedged between postcingular plates 2‴ and 4‴. Plate 1p occupies 30% of the width of the hypotheca. 3) Cells of G. pacificus are 67–77 μm long and 60–76 μm wide in dorsoventral depth, and its surface is smooth. They are identified by the four-sided apical pore plate (Po) surrounded by 30 round pores. A short narrow 1p plate is wedged between the wide postcingular plates 2‴ and 4‴. Plate 1p occupies 20% of the width of the hypotheca. These three newly described species were also characterized by isozyme electrophoresis and DNA sequencing of the D8–D10 region of their large subunit (LSU) rRNA genes. The consistency between species designations based on SEM microscopy and classification inferred from biochemical and genetic heterogeneities was examined among seven isolates of Gambierdiscus. Their classification into four morphospecies was not consistent with groupings inferred from isozyme patterns. Three molecular types could be distinguished based on the comparison of their LSU rDNA sequences. Although G. toxicus TUR was found to be more closely related to G. pacificus, sp. nov. than to other G. toxicus strains, the molecular classification was able to discriminate G. polynesiensis, sp. nov. and G. australes, sp. nov. from G. toxicus. These results suggest the usefulness of the D8–D10 portion of the Gambierdiscus LSU rDNA as a valuable taxonomic marker.     

Systematic revision of the genus Phycodrys (Delesseriaceae,Rhodophyta) from New Zealand,with the descriptions of three new species,P. novae-zelandiae sp. nov., P. franiae sp. nov. and P. adamsiae sp. nov.

Two species of Phycodrys, Phycodrys quercifolia (Bory) Skottsberg and Phycodrys profunda E.Y.Dawson were previously recorded from New Zealand. However, an examination of Phycodrys collections from the New Zealand region showed that all were morphologically different from P. quercifolia (Type locality: the Falkland Islands) and P. profunda (Type locality: CA, USA). RbcL sequence analyses established that the New Zealand Phycodrys species formed a natural assemblage within the genus, consisting of three new species: P. novae-zelandiae sp. nov., P. franiae sp. nov. and P. adamsiae sp. nov. Phycodrys novae-zelandiae is the largest of the three, up to 20 cm in height, with a distinct midrib and multicellular, opposite to subopposite lateral macroscopic veins. It has entirely monostromatic blades except near the midrib and veins, and its procarp contains a three-celled sterile group one (st1) and a one-celled sterile group two (st2). Phycodrys franiae was previously treated as a cryptic species among herbarium collections of P. ‘quercifolia’. It is smaller (4–11 cm high) with weakly developed midribs and veins, the blade is tristromatic throughout, except at the growing margins, and the procarp consists of a four-celled st1 and a two–three-celled st2. Phycodrys adamsiae, previously reported as P. profunda, is a small decumbent or prostrate plant, 1–8 cm long, with a midrib and inconspicuous lateral veins. The blades are tristromatic with serrated margins, two–four-celled surface spines and multicellular marginal holdfasts that differ from those of Californian specimens. The tetrasporangia are borne on marginal bladelets. Phylogenetic analyses place the New Zealand species in a separate group that is distantly removed from most other Phycodrys species.     

Transferring the freshwater dinoflagellate Peridinium wisconsinense (Dinophyceae) to the family Thoracosphaeraceae,with the description of Fusiperidinium gen. nov.

Distinctive spindle‐shaped thecae first described by Samuel Eddy in 1930 and assigned to the genus Peridinium Ehrenberg are commonly reported from freshwater environments in eastern North America. We demonstrate that thecae incubated from cysts of Peridinium wisconsinense Eddy have six cingular plates and a protuberant apical pore complex characteristic of the family Thoracosphaeraceae Schiller 1930 emend. Tangen in Tangen et al . 1982. Small subunit ribosomal DNA (SSU rDNA) and internal transcribed spacer (ITS) sequences confirm the close genetic similarity with Chimonodinium lomnickii (Wo?oszyńska) Craveiro, Calado, Daugbjerg, Gert Hansen & Moestrup and with species recently reassigned to the genus Apocalathium Craveiro, Daugbjerg, Moestrup & Calado that was inferred from previously published LSU rDNA analysis of cysts of P. wisconsinense . Despite sharing identical tabulation with the thoracosphaeracean genera Chimonodinium Craveiro, Calado, Daugbjerg, Gert Hansen & Moestrup and Apocalathium , substantial morphological differences in the morphology of both the thecate and cyst stages of P. wisconsinense led us to reassign this species to the genus Fusiperidinium gen. nov. The phylogenetic position of Fusiperidinium wisconsinense comb. nov., inferred from concatenated data of SSU and LSU sequences, suggests that it evolved from the brackish Scrippsiella lineage, independently of the transition that produced the family Peridiniaceae. Cysts described as Geiselodinium tyonekensis Engelhardt from nonmarine strata from Alaska are apparently identical to the resistant cysts produced by F. wisconsinense . The palynologically‐constrained late Middle Miocene age for the Tyonek Formation provides a minimum age of 11.6 Ma for the evolution of this lineage, coinciding with a rapid glacioeustatic decline in sea level. Our findings also call into question the inclusion of the family Thoracosphaeraceae within the order Peridiniales Haeckel.     

A revision of the genus Cryptonemia (Halymeniaceae,Rhodophyta) in Bermuda,western Atlantic Ocean,including five new species and C. bermudensis (Collins & M. Howe) comb. nov.

Cryptonemia specimens collected in Bermuda over the past two decades were analysed using gene sequences encoding the large subunit of the nuclear ribosomal DNA and the large subunit of RuBisCO as genetic markers to elucidate their phylogenetic positions. They were additionally subjected to morphological assessment and compared with historical collections from the islands. Six species are presently found in the flora including C. bermudensis comb. nov., based on Halymenia bermudensis, and the following five new species: C. abyssalis, C. antricola, C. atrocostalis, C. lacunicola and C. perparva. Of the eight species known in the western Atlantic flora prior to this study, none is found in Bermuda. Specimens reported in the islands in the 1900s attributed to C. crenulata and C. luxurians are representative of the new species, C. antricola and C. atrocostalis, respectively.     

Morphology and taxonomy of five marine sand-dwelling Thecadinium species (Dinophyceae) from Japan, including four new species: Thecadinium arenarium sp. nov., Thecadinium ovatum sp. nov., Thecadinium striatum sp. nov. and Thecadinium yashimaense sp. nov.

Thecadinium inclinatum Balech and four new marine sand‐dwelling species of the dinoflagellate genus Thecadinium are described from the sandy beaches along the coast of Shikoku, Japan. Thecadinium inclinatum is thecate, bilaterally flattened, elliptical in shape, non‐photosynthetic, and measures 55–75 μ in length and 43–59 μ in depth. The epi‐ and hypotheca theca are semielliptical and the thecal surface is smooth with small pores. The plate formula is Po (pore plate), 3′, 7″,?c,?s, 5″′1″′.Thecadinium ovatum sp. nov. is thecate, non‐photosynthetic, bilaterally flattened and almost oval in lateral view. The cell measures 40–50 μm in length and 33–40 μm in depth. The hypotheca has two or three strong antapical spines. The plate formula is 3′, 6″,6c, 5s?, 5″′, 1″′. Thecadinium striatum sp. nov. is thecate, non‐photosynthetic, bilaterally flattened and somewhat elliptical in lateral view. The cell is 33–41 μm long and 23–30 μm deep. Several striae are present on the hypotheca. The plate formula is 3′, 6″, 6c, 5s?, 5″′, 1″″. Thecadinium yashimaense sp. nov. is bilaterally flattened, photosynthetic and elliptical in ventral view. The cell is 44–65 μm long and 23–36 μm wide. The thecal surface is smooth with small pores. he cingulum forms a steep left–handed spiral. The plate formula is Po, 3′, la, 6″, 5c, 4s, 5″′, 1″′. Thecadinium arenarium sp. nov. is somewhat wedge‐shaped in ventral view, photosynthetic with brownish chloroplasts and almost rounded in cross section. The cingulum forms a steep left‐handed spiral. The cell measures 35–41 μm in length and 25–30 μm in width. The thecal surface is weakly reticulated with small pores. The hypotheca is conical. The plate formula is Po, 3′, la, 6″, 5c, 4s, 5″′, 1″″.     

First report of the benthic dinoflagellate,Gambierdiscus belizeanus (Gonyaulacales: Dinophyceae) for the east coast of Sabah,Malaysian Borneo

Species of the genus Gambierdiscus Adachi & Fukuyo, in particular G. toxicus Adachi & Fukuyo are known producers of neurotoxins associated with ciguatera fish poisoning (CFP). In this study live samples were collected from seaweed beds of the east coast of Sabah, Malaysian Borneo and a strain of Gambierdiscus was isolated and cultured. Examination of the thecal fine morphology was undertaken using light, epifluorescence, and scanning electron microscopy. Observed morphological features and their associated morphometric information enabled identification to Gambierdiscus belizeanus Faust. This represents the first report for the occurrence of G. belizeanus in the Asia Pacific region.     

Discovery of a novel type of body scale in the marine dinoflagellate,Amphidinium cupulatisquama sp. nov. (Dinophyceae)

A new species of Amphidinium, A. cupulatisquama Tamura et Horiguchi, from sand samples from Ikei Island, Okinawa Prefecture in subtropical Japan, is described based on light, scanning and transmission electron microscopy and the partial sequencing of the large subunit rDNA gene. The species has a typical morphology for the genus, but is distinguished from previously described species by having a combination of the following characteristics: (i) a relatively large cell (over 30 µm in length); (ii) possessing an eyespot on the dorsal side of the cingulum; (iii) the longitudinal flagellum emerging from a point close to the cingulum; (iv) cell division taking place in the motile phase; and (v) possessing body scales. This is the third species of this genus to possess body scales. The body scales of A. cupulatisquama are uniform and cup‐shaped in side view and elliptical in face view. Their dimensions are 136.4 nm by 91.0 nm by 81.8 nm high. In side view, the scale is seen to have a thick lower half and a thin upper half. This scale type is very different from those of previously reported Amphidinium species (HG114 and HG115). The molecular tree indicated that A. cupulatisquama and the two other strains of body scale‐bearing Amphidinium are distantly related within the Amphidinium clade.     

Pyramidodinium atrofuscum gen. et sp. nov. (Dinophyceae), a new marine sand-dwelling coccoid dinoflagellate from tropical waters

A new marine sand‐dwelling coccoid dinoflagellate Pyramidodinium atrofuscum Horiguchi et Sukigara gen. et sp. nov. is described from Jellyfish Lake, Republic of Palau. The dinoflagellate alternates a non‐motile vegetative stage with a motile gymnodinioid stage within its life cycle. The non‐motile stage is dominant in the life cycle and the dinoflagellate reproduces itself by means of the production of two motile cells. The released motile cell swims only for a short period and is directly transformed into the non‐motile cell. The non‐motile cell is sessile, pyramidal in shape, with a single longitudinal ridge and a double transverse ridge. The surface of the cell wall is covered with many processes. The motile cell has a Gymnodinium‐like morphology, but no apical groove is present. An ultrastructural study revealed that the dinoflagellate possesses typical dinoflagellate organelles. Based on the unique morphology of the vegetative non‐motile stage, we propose a new genus Pyramidodinium for this dinoflagellate, with the type species Pyramidodinium atrofuscum Horiguchi et Sukigara, gen. et sp. nov.     

Cellular and body scale morphology of Heterocapsa huensis sp. nov. (Peridiniales, Dinophyceae) found in Hue, Vietnam

Cellular and body scale structure of a new armored dinoflagellate Heterocapsa huensis , collected from Hue, Vietnam were investigated. Morphology of motile cell was observed by light, fluorescent and scanning electron microscopy, and body scale structure was examined by whole mounts of transmission electron microscopy. Cells of H. huensis were ellipsoid with a spherical nucleus located in the posterior and multiple pyrenoids located above the nucleus; this arrangement was similar to that of Heterocapsa pygmaea . Transmission electron microscopy revealed ultrastructure of the body scales consisted of a rounded triangular basal plate and three-dimensional ornaments. Structure of the basal plate resembles that of Heterocapsa illdefina ; however, the number of the peripheral spine is different from that of H. illdefina and this structure has never been reported from Heterocapsa species. A new Heterocapsa species, H. huensis Iwataki et Matsuoka sp. nov., is described based on positions of organelles and body scale ultrastructure.     

Polysiphonia freshwateri sp. nov. and Polysiphonia koreana sp. nov.: two new species of Polysiphonia (Rhodomelaceae,Rhodophyta) from Korea

Polysiphonia sensu lato comprises approximately 200 species, which are currently assigned to several different genera. To date, one of these genera, namely, Polysiphonia, has been reported to have 17 species. Here, we describe for the first time P. freshwateri sp. nov. and P. koreana sp. nov. from Uljin and Ulleung Island, Korea, based on morphological and molecular evidence. Polysiphonia freshwateri sp. nov. and P. koreana sp. nov. are characterized by having the typical Polysiphonia features. Polysiphonia freshwateri sp. nov. is further characterized by having abundant trichoblasts, conspicuous scar cells, and tetrasporangia arranged in spiral series. Polysiphonia koreana sp. nov. is further characterized by having very scarce scar cells placed between two pericentral cells, from which cicatrigenous branches arise. The results of our rbcL sequence analyses support the taxonomic placement of P. freshwateri sp. nov. and P. koreana sp. nov. within Polysiphonia.     

Ailadinium reticulatum gen. et sp. nov. (Dinophyceae), a New Thecate,Marine, Sand‐Dwelling Dinoflagellate from the Northern Red Sea

A new photosynthetic, sand‐dwelling marine dinoflagellate, Ailadinium reticulatum gen. et sp. nov., is described from the Jordanian coast in the Gulf of Aqaba, northern Red Sea, based on detailed morphological and molecular data. A. reticulatum is a large (53–61 μm long and 38–48 μm wide), dorsoventrally compressed species, with the epitheca smaller than the hypotheca. The theca of this new species is thick and peculiarly ornamented with round to polygonal depressions forming a foveate‐reticulate thecal surface structure. The Kofoidian thecal tabulation is APC (Po, cp), 4′, 2a, 6′′, 6c, 4s, 6′′′, 1p, 1′′′′ or alternatively it can be interpreted as APC, 4′, 2a, 6′′, 6c, 4s, 6′′′, 2′′′′. The plate pattern of A. reticulatum is noticeably different from described dinoflagellate genera. Phylogenetic analyses based on the SSU and LSU rDNA genes did not show any supported affinities with currently known thecate dinoflagellates.