Apical Structure Complex Research Articles

Morphology and molecular phylogeny of Bindiferia gen. nov. (Dinophyceae), a new marine, sand-dwelling dinoflagellate genus formerly classified within Amphidinium

ABSTRACT As historically described, the genus Amphidinum has long been recognized to be polyphyletic. Amphidinium sensu lato is the most diverse of all marine benthic dinoflagellate genera and after the redefinition of the sensu stricto genus about 100 species remained of unknown generic affiliation. A species similar to the Australian sensu lato species Amphidinium boggayum was discovered in France. Morphological and molecular phylogenetic data supported the description of a new species and the classification of the two closely related species within a new athecate, photosynthetic, benthic genus, named Bindiferia. Cells were dorsoventrally flattened with smaller asymmetric epicone. The cingulum was descending. The sulcus reached the antapex and had an extension onto the epicone. The apical structure complex formed an anticlockwise loop around the apex. A red stigma was located in the anterior part of the epicone, near the apex. Vegetative division occurred in a non-motile stage within a hyaline sheath. Non-motile cells had dorsal hypocone grooves. The two species differed from each other in epicone size, cingulum displacement, nucleus location and surface groove occurrence. The molecular phylogenetic results showed that Bindiferia is a distinct taxon that is only distantly related to Amphidinium sensu stricto. The nearest sister group to Bindiferia could not be reliably determined, but the apical structure complex suggested a close relationship to the genera of the Gymnodinium sensu stricto clade.

Morpho-molecular description of a new HAB species, Pseudocochlodinium profundisulcus gen. et sp. nov., and its LSU rRNA gene based genetic diversity and geographical distribution

Harmful algal blooms (HABs) caused by an unknown dinoflagellate species have frequently occurred in the Pearl River Estuary, China Since 2006. These blooms were associated with severe water discoloration and economic losses, ranging from several km2 to 300 km2 with the maximum recorded cell density being 2.77 × 107 cells⋅L−1. This unknown dinoflagellate species was initially identified as Cochlodinium geminatum and subsequently reclassified as Polykrikos geminatus. However, after reviewing the original descriptions for Cochlodinium geminatum sensu Schütt (1895) and the genus Polykrikos, we considered this species is incongruent with their original descriptions. Further morphological examinations and particularly phylogenetic analyses based on the SSU and partial LSU rRNA genes of isolates and resting cysts from China and Japan prompted us to consider it a new species of a new genus. This new species was proposed to be Pseudocochlodinium profundisulcus gen. et sp. nov., based on its open comma-shaped apical structure complex (ASC), cingulum encircling the cell less than one and a half turns, a deep sulcus with a torsion of a half turn, either single cell or cell chain consisting of two cells with the same number of nuclei and zooids, the resting cyst bearing lobed ornaments, and the evolutionary distances from Polykrikos (and others) on the phylogenetic trees constructed using the concatenated SSU and partial LSU rRNA gene sequences. Metabarcoding investigation of surface sediment samples collected in China revealed that the species to be widely present along the entire Chinese coast with the highest abundance in the South China Sea. Further re-analysis of the Tara Oceans metabarcoding dataset targeting the SSU rRNA gene V9 domain suggested a global distribution of this new genus. Phylogenetic analyses on 46 OTUs (average length: ∼552 bases) of its LSU rRNA gene sequences (mainly D1-D2 domains) obtained from surface sediment samples revealed intraspecific genetic diversity of this species. Interestingly, based on the different distributions and the abundance of these OTUs along the coast of China, this species appeared to have expanded its distribution from the South China Sea to the northern Yellow Sea, or preferred a warm water habitat. We consider that the present work improves the taxonomy and provides important insights into the biogeography of Pseudocochlodinium profundisulcus.⋅

Morphology, ultrastructure, and molecular phylogeny of the unarmoured dinoflagellate Kirithra sigma sp. nov. (Ceratoperidiniaceae, Dinophyceae)

ABSTRACT A Gymnodinium-like dinoflagellate, Kirithra sigma sp. nov., was isolated from Jiaozhou Bay, China, and the southern coast of Korea, and examined by light, scanning, and transmission electron microscopy. Pigment composition and nuclear-encoded partial large subunit ribosomal RNA (LSU rRNA) gene were determined. Cells were oval-shaped (26.4‒36.4 μm long and 17.7‒27.2 μm wide), with a conical epicone and hemispherical hypocone. Its cingulum and sulcus were deep and wide. The cingulum encircled the cell completely, its ends slightly overlapping and displaced one to two cingular widths; the sulcus and sulcal extension formed a sigmoid curve. The anterior sulcal extension contacted the apical structure complex (ASC). The ASC formed a complete circle, consisting of three parallel series of vesicles, and the amphiesmal vesicles within ASC were arranged with one central vesicle (sometimes two) encircled by approximately three rows of polygonal amphiesmal vesicles. Polygonal amphiesmal vesicles of different sizes covered the whole cell surface. The subspherical nucleus was located in the hypocone. Pigment analysis showed that peridinin was the main accessory pigment. The present species is morphologically similar to Kirithra asteri but differs in numbers and rows of amphiesmal vesicles within the ASC, absence of ventral pore, and a sigmoid curve formed by the sulcus and sulcal extension. The partial LSU rRNA gene sequence (c. 1400 bases) of K. sigma differed by c. 5.3% from that of K. asteri. Molecular phylogeny revealed that K. sigma formed an independent sister clade to K. asteri, indicating they are congeneric but different species.

The Harmful Unarmored Dinoflagellate Karlodinium in Japan and Philippines, with Reference to Ultrastructure and Micropredation of Karlodinium azanzae sp. nov. (Kareniaceae, Dinophyceae)1.

In all, 26 cultures of the harmful marine dinoflagellate Karlodinium, isolated from Japanese and Philippine coastal waters, were examined using LM, SEM, and molecular phylogeny inferred from ITS and LSU rDNA. Seven Karlodinium species (six from Japan and four from Philippines), K.australe, K.ballantinum, K.decipiens, K.gentienii, K.veneficum, K.zhouanum, and a novel species Karlodinium azanzae sp. nov., were identified based on their morphology and phylogenetic positions. Karlodinium azanzae from Manila Bay, Philippines was further characterized by TEM, HPLC (chloroplast pigment), and bioassay on brine shrimp and other marine zooplankton. Cells of K.azanzae were the largest (mean 25.3µm long) in Karlodinium, possessed numerous tiny reflective particles, starch grains, and lipid granules, and usually swam at the bottom of the culture vessel. The straight apical structure complex and a ventral pore were common to the genus. The longitudinally elongated nucleus was located at the center, and the yellowish chloroplasts contained an embedded pyrenoid and carotenoid pigments typical of the genus (i.e., fucoxanthin as major carotenoid with its derivatives). TEM revealed a part of the flagellar apparatus, of which the long striated ventral connective is the first report in the Kareniaceae. Phylogenetic trees showed closest affinity of K.azanzae with K.australe and K.armiger. The new species could be differentiated from related species by cell size, position of the nucleus, and characteristic swimming behavior. Lethality of K.azanzae to large zooplankton and micropredation using a developed peduncle was also observed.

Reclassification of Gyrodinium flavescens Kofoid & Swezy as Torquentidium flavescens comb. nov. (Ceratoperidiniaceae, Dinophyceae), based on morphology and phylogeny

ABSTRACTA Cochlodinium-like species was isolated from the Korean coast and examined by light microscopy and scanning electron microscopy (SEM). The species was characterised by cellular torsion, cingulum with 1.25 turns and sulcus with 0.25 turn, and a hemispherical hypocone. The nucleus was curved, and sausage-shaped, and there were small, dark grains distributed at the cell periphery, which were also reported in the original description of Gyrodinium flavescens by Kofoid & Swezy (1921). SEM observations revealed that the apical structure complex of the species formed a complete circle surrounding the cell apex, consisting of three parallel rows of vesicles; and that the outer rows, composed of small vesicles, were visible along the sides of cingulum. These morphological features were identical to those of Torquentidium convolutum. Phylogeny-based on small subunit and large subunit rDNA sequences, confirmed that the species was closely related to T. convolutum within the family Ceratoperidiniaceae. However, the species was distinct from T. convolutum by differences in cingulum and sulcus turn numbers, shape of hypocone in dorsal view and size. Based on morphological and phylogenetic analyses, we propose Torquentidium flavescens comb. nov. nested within the family Ceratoperidiniaceae.

Clarifying the taxonomy of Gymnodinium fuscum var. rubrum from Bavaria (Germany) and placing it in a molecular phylogeny of the Gymnodiniaceae (Dinophyceae)

The taxonomy of Gymnodinium and the Gymnodiniaceae is inconsistent, because the systematic position of the type species, G. fuscum, is elusive at present. Historical names of microscopic species are frequently ambiguous, making reliable determination difficult, although it is crucial to fully explore the biology of the organisms. We collected material at the type locality of a historical variety, namely Gymnodinium fuscum var. rubrum, var. nov., and established a strain for morphological and molecular studies. The motile cells showed a characteristically obovate shape in outline, with acute antapex, and a horseshoe-shaped apical structure complex, which is characteristic for many Gymnodiniaceae. Older cultivated material further exhibited red granules in the cell, being the name-giving diagnostic character of the variety. In a molecular phylogeny, monophyletic Gymnodiniaceae segregated into seven clades at high taxonomic level. The phylogenetic resolution of the molecular DNA-tree provides evidence for a dynamic evolutionary scenario of Gymnodiniaceae and might prove helpful for an improved classification of the group. As the taxonomic result, we validate and epitypify the historical name, G. fuscum var. rubrum, var. nov., showing diagnostic traits such as a descending (but not straight) cingulum and a reciprocal size ratio between episome and hyposome in comparison to the regular form of G. fuscum.

Dactylodinium arachnoides sp. nov. (Borghiellaceae, Dinophyceae): a new marine dinoflagellate with a loop-shaped apical structure complex and tubular membranous extrusomes

ABSTRACTThe morphology and phylogeny of an enigmatic marine dinoflagellate were examined using LM, SEM, TEM, and molecular phylogeny inferred from rDNA. Cells were elliptical with a conical epicone and a hemispherical hypocone. They measured c. 24.7 µm long, with a cingulum descending approximately twice its own width. Amphiesmal vesicles were thick and transparent, and were supported by translucent ridges at the suture, which were distinctive and visible by light microscopy. A large peduncle extending to the anterior was accommodated in the anterior sulcal extension. A nucleus was usually located in the hypocone, and chloroplasts were distributed peripherally. The apical structure complex was loop-shaped, originating from the right side of the anterior sulcal extension and extending upward to encircle the dorsal side of the cell apex in an anticlockwise direction. TEM showed trichocysts with lateral hairs, a pyrenoid penetrated by cytoplasmic insertions containing tubules, and an eyespot comprising globules within the chloroplast. Cells also had tubular extrusomes, unique in dinoflagellates, composed of a membrane stacked in a vesicle, and formed a long membranous tube with 2–4 finger-like terminal branches after being released from the vesicle. Molecular phylogenies showed the affinity of the species to Dactylodinium pterobelotum in the family Borghiellaceae. We propose Dactylodinium arachnoides sp. nov. for the dinoflagellate described here. It shares the characteristic traits of amphiesmal vesicles, a peduncle, a pyrenoid, and trichocysts with D. pterobelotum, whereas the number of amphiesmal vesicles, the loop-shaped apical structure complex, eyespot type A, and tubular membranous extrusomes are distinct.

Gertia stigmatica gen. et sp. nov. (Kareniaceae, Dinophyceae), a New Marine Unarmored Dinoflagellate Possessing the Peridinin-type Chloroplast with an Eyespot

Marine unarmored dinoflagellates in the family Kareniaceae are known to possess chloroplasts of haptophyte origin, which contain fucoxanthin and its derivatives as major carotenoids, and lack peridinin. In the present study, the first species with the peridinin-type chloroplast in this family, Gertia stigmatica gen. et sp. nov., is described on the basis of ultrastructure, photosynthetic pigment composition, and molecular phylogeny inferred from nucleus- and chloroplast-encoded genes. Cells of G. stigmatica were small and harboring a chloroplast with an eyespot and two pyrenoids. The apical structure complex was straight, similar to Karenia and Karlodinium. Under transmission electron microscopy, the chloroplast was surrounded by two membranes, and the eyespot was composed of a single layer of osmiophilic globules (eyespot type A); this was never previously reported from the Kareniaceae. High performance liquid chromatography demonstrated the chloroplast contains peridinin, and neither fucoxanthin nor 19'-acyloxyfucoxanthins was identified. A phylogeny based on nucleus-encoded rDNAs suggested a position of G. stigmatica in the Kareniaceae, but not clustered within the previously described genera, i.e., Karenia, Karlodinium and Takayama. A phylogeny of chloroplast-encoded psbA, psbC and psbD indicated the chloroplast is of peridinin-type typical of dinoflagellates, but the most related species remains unclear.

Morphological variation, ultrastructure, pigment composition and phylogeny of the star-shaped dinoflagellate Asterodinium gracile (Kareniaceae, Dinophyceae)

ABSTRACTMorphological variation, ultrastructure, pigment composition and phylogeny of an oceanic, star-shaped, unarmored dinoflagellate Asterodinium gracile were examined using a culture established from Nagasaki, Japan, in 2017. Our examinations were based on LM, SEM, TEM, high-performance liquid chromatography (HPLC) and molecular phylogeny inferred from nuclear-encoded internal transcribed spacer (ITS) and 28S rDNA and chloroplast-encoded 16S rDNA sequences. Typical cells were stellate, possessing an apical, two lateral, and two antapical extensions. Cells ranged in size from 62.5 to 105.6 µm long and 21.2 to 39.5 µm in cingular width. The length and orientation of cellular extensions were variable; that is, two lateral extensions were usually symmetrically bent or retracted. Ellipsoidal cells without extensions were also observed. The apical structure complex was straight or slightly sigmoid. A spherical nucleus was located at the left side in the hyposome. The numerous discoid, yellowish-brown chloroplasts were variable in size and distributed also in the cell extensions. Among Kareniaceae, the lack of a pyrenoid in Asterodinium is unique. Nuclear-encoded ITS and 28S phylogenies suggested that A. gracile is closely related to Karenia papilionacea in the Kareniaceae, but its affinity to Brachidinium capitatum, another species with extensions, was unclear. A phylogeny of 16S rDNA showed that chloroplasts of A. gracile are related to other kareniaceans derived from a haptophyte. These chloroplasts were of the fucoxanthin type, composed of pigments similar to those in other Kareniaceae; however, the detection of 19′-hexanoyloxyfucoxanthin as a major carotenoid, the low amount of fucoxanthin, and lack of 19′-butanoyloxyfucoxanthin in A. gracile were unusual for the family.

Characterization of the unarmored dinoflagellate Pseliodinium pirum (Ceratoperidiniaceae) from Jiaozhou Bay, China

SUMMARYA strain of Pseliodinium pirum was isolated from Jiaozhou Bay, China, identified based on a recently emended classification, and further characterized for its morphology using light, scanning electron, and transmission electron microscopy, and phylogeny based on SSU and partial LSU rDNA sequences. The pigment composition, life history, and potential effects on aquatic animals were also examined. We observed the typical characteristics of Ceratoperidiniaceae, in which its apical structure complex (ASC) formed a circular loop, and the ASC of our isolate comprised four to five rows of vesicles, and connected with sulcal intrusion. Epifluorescence and transmission electron microscopy revealed a bean‐shaped, centrally located nucleus, with at least 76 chromosomes. Numerous rod‐shaped chloroplasts were in connection to the irregularly shaped pyrenoids. Pigment analysis showed that peridinin was the most abundant among all carotenoids and other pigments. Phylogenetic analyses using maximum likelihood and Bayesian inference indicated that our isolate is conspecific with the entity Cochlodinium cf. helix (accession No. KF245459), but different from Ceratoperidinium, Kirithra, and other unidentified species of the family Ceratoperidiniaceae. Pseliodinium pirum could produce sexual, thin‐walled cyst, with subspherical and spherical shape and smooth surface (without spines or rough projections). The cyst could germinate within 3 days. Bioassays did not show adverse effects of P. pirum on the finfish Oryzias melastigma and the rotifer Brachionus plicatilis, indicating it may not be a harmful species.

Gyrodinium jinhaense n. sp., a New Heterotrophic Unarmored Dinoflagellate from the Coastal Waters of Korea.

Four unarmored heterotrophic dinoflagellates were isolated from the coastal waters of southern Korea. The rDNA sequences of four clonal cultures were determined, and the morphology of one of the four strains was examined using light and scanning and transmission electron microscopy. The large subunit (LSU) and small subunit (SSU) rDNA sequences of each of the strains differed by 0-0.9% from those of the other strains, and the SSU rDNA sequence of the strain differed by 1.8-4.4% from those of other Gyrodinium species, whereas the LSU (D1-D2) rDNA sequence differed by 12.4-22.2%. Furthermore, phylogenetic trees showed that Gyrodinium jinhaense n. sp. formed a distinctive clade among the other Gyrodinium species. Meanwhile, microscopy revealed an elliptical bisected apical structure complex and a cingulum that was displaced by approximately one-quarter of the cell length, which confirmed that the dinoflagellate belonged to the genus Gyrodinium. However, the cell surface was ornamented with 16 longitudinal striations, both on the episome and hyposome, unlike other Gyrodinium species. Furthermore, the cells were observed to have pusule systems and trichocysts but lacked mucocysts. Based on morphology and molecular data, we consider this strain to be a new species in the genus Gyrodinium and thus, propose that it be assigned to the name G.jinhaense n. sp.

Molecular phylogeny and morphology of Torquentidium gen. et comb. nov. for Cochlodinium convolutum and allied species (Ceratoperidiniaceae, Dinophyceae)

ABSTRACT Although the genus Pseliodinium within the family Ceratoperidiniaceae includes Ceratoperidinium falcatum, Cochlodinium cf. convolutum, C. cf. helix and unidentified Cochlodinium species, it is in need of reclassification because of the morphological differences and lack of morphological details of the species within the genus. A Cochlodinium-like species was isolated from a Korean coastal area and examined by light and scanning electron microscopy, as well as by the analysis of nuclear-encoded small subunit (SSU) and large subunit (LSU) rDNA. The Korean isolate was morphologically characterized by cellular torsion, and outer rows consisting of small rectangular and polygonal vesicles were visible along the sides of the cingulum. The apical structure complex (ASC) formed a complete circle surrounding the cell apex, consisting of three parallel rows of vesicles. In the ASC, the upper row was sometimes hidden and very small knobs were present on the central row. Morphological characteristics separated the Korean isolate from Ce. margalefii, Pseliodinium fusus(=Ce. falcatum), Kirithra asteri and Gymnodinium species (Gymnodinium sp. 1 and sp. 2) within the family Ceratoperidiniaceae. However, the morphological characteristics coincided with those of the previously recorded C. convolutum, C. cf. helix and other Cochlodinium species within the same family. Phylogeny based on SSU and LSU rDNA sequences revealed that the Korean isolate forms a clade independent from P. fusus, although they have high sequence similarity. Based on the morphological and phylogenetic analyses, we propose the new genus Torquentidium gen. nov. and a new combination for C. cf. helix and the Korean isolate Torquentidium convolutum erected within the family Ceratoperidiniaceae.

Morphology, ultrastructure, and molecular phylogeny of Wangodinium sinense gen. et sp. nov. (Gymnodiniales, Dinophyceae) and revisiting of Gymnodinium dorsalisulcum and Gymnodinium impudicum.

The genus Gymnodinium includes many morphologically similar species, but molecular phylogenies show that it is polyphyletic. Eight strains of Gymnodinium impudicum, Gymnodinium dorsalisulcum and a novel Gymnodinium-like species from Chinese and Malaysian waters and the Mediterranean Sea were established. All of these strains were examined with light microscopy, scanning electron microscopy and transmission electron microscopy. SSU, LSU and internal transcribed spacers rDNA sequences were obtained. A new genus, Wangodinium, was erected to incorporate strains with a loop-shaped apical structure complex (ASC) comprising two rows of amphiesmal vesicles, here referred to as a new type of ASC. The chloroplasts of Wangodinium sinense are enveloped by two membranes. Pigment analysis shows that peridinin is the main accessory pigment in W.sinense. Wangodinium differs from other genera mainly in its unique ASC, and additionally differs from Gymnodinium in the absence of nuclear chambers, and from Lepidodinium in the absence of Chl b and nuclear chambers. New morphological information was provided for G.dorsalisulcum and G.impudicum, e.g., a short sulcal intrusion in G.dorsalisulcum; nuclear chambers in G.impudicum and G.dorsalisulcum; and a chloroplast enveloped by two membranes in G.impudicum. Molecular phylogeny was inferred using maximum likelihood and Bayesian inference with independent SSU and LSU rDNA sequences. Our results support the classification of Wangodinium within the Gymnodiniales sensu stricto clade and it is close to Lepidodinium. Our results also support the close relationship among G.dorsalisulcum, G.impudicum, and Barrufeta. Further research is needed to assign these Gymnodinium species to Barrufeta or to erect new genera.

Ultrastructure and Phylogeny of Kirithra asteri gen. et sp. nov. (Ceratoperidiniaceae, Dinophyceae) — a Free-Living, Thin-Walled Marine Photosynthetic Dinoflagellate from Argentina

A gymnodinioid photosynthetic dinoflagellate was isolated from Argentina and examined by light and electron microscopy and analysis of nuclear-encoded LSU rDNA. Kirithra asteri gen. et sp. nov. was proposed as morphology and molecular phylogeny separated this dinoflagellate from others within the family Ceratoperidiniaceae. Cells were surrounded by a hyaline amphiesma comprising polygonal vesicles. Each vesicle contained a honeycomb and a trilaminar structure. An anterior sulcal extension ending in a complete circle formed the apical structure complex (ASC), which characterizes Ceratoperidiniaceae. The ASC comprised three rows of vesicles. The nucleus was located in the hypocone, and several large, irregularly shaped vesicles were present in the epi- and hypocone. Chloroplasts were surrounded by three membranes, and grana-like arrangements of thylakoids were observed in one strain used for ultrastructural study. The cell centre contained 1-3 multiple-stalked pyrenoids and membrane-bound vesicles containing tile-like structures surrounded each pyrenoid. Two pusules with collecting chambers and associated vesicles branched off each of the flagellar canals. The flagellar apparatus featured a ventral connective between the amphiesma and the R1 root, and almost opposite basal bodies, rarely seen in dinoflagellates. This was the first ultrastructural study of a species within Ceratoperidiniaceae.

Cyst‐motile stage relationship and molecular phylogeny of a new freshwater dinoflagellate Gymnodinium plasticum from Plastic Lake, Canada

SUMMARYThe dinophyceaen genus Gymnodinium was established with the freshwater species G. fuscum as type. According to Thessen et al. (2012), there are 268 species, with the majority marine species. In recently published molecular phylogenies based on ribosomal DNA sequences, Gymnodinium is polyphyletic. Here, a new freshwater Gymnodinium species, G. plasticum, is described from Plastic Lake, Ontario, Canada. Two strains were established by incubating single cysts, and their morphology was examined with light microscopy and scanning electron microscopy. The cyst had a rounded epicyst and hypocyst with a wide cingulum and smooth surface. Vegetative cells were characterized by an elongated nucleus running vertically and a deep sulcal intrusion. The apical structure complex was horseshoe‐shaped and consisted of two pronounced ridges with a deep internal groove, encircling 80% of the apex. Small subunit ribosomal DNA (SSU rDNA), large subunit ribosomal DNA (LSU rDNA) and internal transcribed spacer (ITS) sequences were obtained from cultured strains. Molecular phylogeny based on concatenated SSU, LSU and ITS sequences supports the monophyly of the Gymnodiniales sensu stricto clade but our results suggest that many Gymnodinium species might need reclassification. Gymnodinium plasticum is closest to Dissodinium pseudolunula in our phylogeny but distant from the type species G. fuscum, as are the other gymnodiniacean taxa.

Cryptic diversity within the harmful dinoflagellate Akashiwo sanguinea in coastal Chinese waters is related to differentiated ecological niches

Blooms of the harmful dinoflagellate Akashiwo sanguinea are responsible for the mass mortality of fish and invertebrates in coastal waters. This cosmopolitan species includes several genetically differentiated clades. Four clonal cultures were established by isolating single cells from Xiamen Harbour (the East China Sea) for morphological and genetic analyses. The cultures displayed identical morphology but were genetically different, thus revealing presence of cryptic diversity in the study area. New details of the apical structure complex of Akashiwo sanguinea were also found. To investigate whether the observed cryptic diversity was related to environmental differentiation, 634 cells were obtained from seasonal water samples collected from 2008 to 2012. These cells were sequenced by single-cell PCR. For comparison with Chinese material, additional large subunit ribosomal DNA sequences were obtained for three established strains from Malaysian and French waters. To examine potential ecological differentiation of the distinct genotypes, growth responses of the studied strains were tested under laboratory conditions at temperatures of 12°C to 33°C. These experiments showed four distinct ribotypes of A. sanguinea globally, with the ribotypes A and B co-occuring in Xiamen Harbour. Ribotype A of A. sanguinea was present year-round in Xiamen Harbour, but it only bloomed in the winter and spring, thus corresponding to the winter type. In contrast, A. sanguinea ribotype B bloomed only in the summer, corresponding to the summer type. This differentiation supports the temperature optimum conditions that were established for these two ribotypes in the laboratory. Ribotype A grew better at lower temperatures compared to ribotype B which preferred higher temperatures. These findings support the idea that various ribotypes of A. sanguinea correspond to distinct ecotypes and allopatric speciation occurred in different climatic regions followed by dispersal.

Katodinium glaucum (Dinophyceae) revisited: proposal of new genus, family and order based on ultrastructure and phylogeny

:Since its description in 1917, the taxonomy of Katodinium glaucum, a cosmopolitan heterotrophic marine dinoflagellate, has been in a state of flux. We added to this by arguing that Kat. glaucum is synonymous with both Amphidinium extensum and Gymnodinium vestifici. We presented a brief historical account of its shifting positions in Katodinium and Gyrodinium and a comparison with Gym. vestifici and A. extensum. The conclusions from our experimental work on a Kat. glaucum culture were based on both pheno- and genotypic characters obtained by light and electron microscopy and DNA sequencing. A nuclear-encoded large-subunit rDNA-based phylogeny revealed that Kat. glaucum and species of Torodinium are closely related and form a highly supported monophyletic lineage. The ultrastructural examination showed the apical structure complex of Kat. glaucum to comprise three rows of amphiesmal vesicles located beneath the cap-like projection on the anterior part of the cell. Torodinium, in comparison, possessed a counterclockwise spiral apical structure complex positioned on top of its bill-like apical projection. Species of both genera were longitudinally striated. The type species of Katodinium, Kat. nieuportense, was illustrated without any apical structures and without longitudinal striations, and because both morphological and molecular analysis also rejected relationship to Amphidinium, Gymnodinium and Gyrodinium, we proposed to erect a new genus for this species, Kapelodinium gen. nov. As the species name vestifici had priority over glaucum, the new name for the species was Kapelodinium vestifici comb. nov. To accommodate for the distinct difference in morphology of the apical structure complex in Torodinium and Kapelodinium, we further proposed two new monotypic families, Torodiniaceae fam. nov. and Kapelodiniaceae fam. nov. of the order Torodiniales ord. nov., which in the molecular trees was not closely related to other dinoflagellates. The ultrastructural study of Kap. vestifici gen. & comb. nov. also revealed a number of characteristic features, such as a novel lip-like suture within the sulcus, cross-striated filaments beneath the apical projection and atypical nuclear chambers with pores towards both the nucleoplasm and the cytoplasm.

Cyst-motile stage relationship, morphology, ultrastructure, and molecular phylogeny of the gymnodinioid dinoflagellateBarrufeta resplendenscomb. nov., formerly known asGyrodinium resplendens, isolated from the Gulf of Mexico

In the present study, we redescribed Gyrodinium resplendens through incubation of process bearing cysts extracted from sediment collected in the northern Gulf of Mexico. The morphology and ultrastructure of the motile stage and cyst stage were examined using light microscopy, scanning electron microscopy, and transmission electron microscopy and this revealed that the species should be transferred to the genus Barrufeta. This genus differs from other gymnodinioid genera in possessing a Smurf-cap apical structure complex (ASC) and currently encompasses only one species, Barrufeta bravensis. B.resplendens shows a Smurf-cap ASC that consists of three rows of elongated vesicles with small knobs in the middle one. B.resplendens is very similar to B.bravensis in cell morphology, but can be separated using the ultrastructure such as the shape and location of nucleus and pyrenoids, which highlights the importance of ultrastructure at inter-specific level in the genus Barrufeta. The unique cysts of B.resplendens are brown and process bearing, and have a tremic archeopyle with a zigzag margin on the dorsal side of the epicyst, and not polar as in cysts of Polykrikos. The cysts do not survive the palynological treatment used here and probably have a wide distribution. Maximum-likelihood and Bayesian inference were carried out based on partial large subunit ribosomal DNA (LSU rDNA) sequences. Molecular phylogeny supports that the genus Barrufeta is monophyletic, and that the genus Gymnodinium is polyphyletic. Our results suggest that details of the ASC together with ultrastructure are potential features to subdivide the genus Gymnodinium.

Studies on woloszynskioid dinoflagellates VII. Description of Borghiella andersenii sp. nov.: light and electron microscopy and phylogeny based on LSU rDNA

Freshwater woloszynskioid dinoflagellates were collected independently in Scotland and Portugal and found to belong to a previously unknown species of the genus Borghiella, here described as B. andersenii. The new species differs in morphology and nuclear-encoded LSU rDNA and ITS sequences from B. dodgei and B. tenuissima, the two species presently comprising the genus Borghiella. Unusual features of the new species were observed particularly during asexual reproduction, which took place in the motile stage – as in many other dinoflagellates – or in a so-called division cyst, recalling cell division in the family Tovelliaceae. Such diversity in cell division is rarely reported in dinoflagellates. Morphologically Borghiella andersenii differs from B. tenuissima in being only slightly compressed dorsoventrally whereas the latter species is flat. The slight compression is also visible in lateral view. Borghiella andersenii and B. dodgei are more challenging to discriminate but the apical structure complex is only half the length in B. andersenii compared with B. dodgei (3–4 vs 6 µm). This difference can only be accounted for in the scanning electron microscope. At the light microscopy level the epicone in B. andersenii is rounded whereas it is conical in B. dodgei. Sexual reproduction in Borghiella andersenii was homothallic by formation of planozygotes, followed by apparent resting cysts. Phylogenetic studies on woloszynskioids have recently shown that they comprise a polyphyletic assemblage, which has been divided into the three families Borghiellaceae, Tovelliaceae and Suessiaceae. New species of the three families are now being found rapidly in many parts of the world, proving that the techniques required to investigate these small, morphologically similar dinoflagellates are now in place and proving that such ‘gymnodinioids’ or ‘woloszynskioids’ comprise an often overlooked biological entity in both marine and freshwater biotopes. Based on LSU rDNA, B. andersenii is most closely related to B. tenuissima.

On Levanderina fissa gen. & comb. nov. (Dinophyceae) (syn. Gymnodinium fissum, Gyrodinium instriatum, Gyr. uncatenum), a dinoflagellate with a very unusual sulcus

:Gymnodinium fissum was described by Levander in 1894 from the Baltic Sea near Helsinki, and we argue, on the basis of morphological and molecular studies of material from the type locality, and on cultures from the Åland islands, Puerto Rico, Portugal and United States identified as Gyrodinium instriatum, Gymnodinium instriatum, Gyrodinium uncatenum and Gyrodinium sp., that all these taxa are conspecific. They are morphologically and genetically distinct from Gymnodinium and are described here as Levanderina fissa gen. & comb. nov. This species also includes Gyrodinium pavillardii. Levander observed chloroplasts in the cell and on some occasions diatoms, probably the first report of mixotrophy in a dinoflagellate. Biecheler in 1952 described the process of food uptake in Gyr. pavillardii, feeding it with ciliates and other dinoflagellates. Prey was taken up through the posterior part of the sulcus, some prey items being almost as large as the host. Our observations showed that the longitudinal flagellum, in contrast to what has been described in all other dinoflagellates possessing a longitudinal sulcal furrow, is not located in the furrow but in a separate, internal tube beneath the sulcal furrow. The tube opened to the exterior dorsally near the posterior end of the cell, and the sulcus appeared to be used for food uptake only. The cytoskeleton of L. fissa was complex and included a large number of muscle-like fibres. Food uptake using the sulcus involved major changes of cell shape, which requires the presence of a highly flexible cytoskeleton. Levanderina fissa was not morphologically or genetically close to any other dinoflagellate for which molecular sequences were available. The detailed structure of the apical furrow or acrobase comprised three rows of elongate vesicles, one row forming the bottom of a furrow. The new term apical structure complex (ASC) is introduced as a general term to replace apical furrow or acrobase, none of which adequately describes all the many known types. The ASC in Levanderina may be characteristic of most if not all species of the Gymnodiniales (an apomorphy of the order?) and different from the types present in the Suessiales, the other order of mainly thin-walled dinoflagellates.