一株淡水多甲藻的形态观察和多甲藻属系统发育分析

通过形态学方法将中国淡水藻种库一株编号为FACHB-329的甲藻鉴定为楯形多甲藻不等变种Peridinium umbonatum var.inaequale Lemmermann,并且描述了此甲藻所产生的孢囊形态。还对该藻的SSU和LSU rDNA序列进行测序,系统发育分析的结果也支持形态鉴定。系统发育研究结果表明多甲藻属是多系起源的,本研究所用的多甲藻属类群可以形成两个大的分枝——Peridinium umbonatum类群形成的分枝和狭义多甲藻属类群（Peridinium sensu stricto）形成的分枝。并且形态学和分子数据都显示了P.umbonatum类群与狭义多甲藻属类群之间的差异。P.umbonatum类群形成的淡水单系类群虽然没有高度的自展支持但获得了不同的系统发育分析的支持,而狭义多甲藻属类群形成的单系类群获得了高度的支持。淡水多甲藻P.aciculiferum和P.wierzejskii则与海洋斯氏藻属关系密切。     

ACQUIRING SCANNING ELECTRON MICROSCOPICAL,LIGHT MICROSCOPICAL AND MULTIPLE GENE SEQUENCE DATA FROM A SINGLE DINOFLAGELLATE CELL1

We have developed a useful method to obtain light and scanning electron micrographs of a single dinoflagellate cell, prior to applying the cell to the single cell PCR technique. This method allows us to record detailed morphological information on any cell used for sequencing, which can be extremely important for the future identification of the organism, because cells used for single cell PCR usually cannot be retained. Furthermore, by applying multiple sets of PCR primers at the same time, we have successfully amplified and sequenced multiple genes (and DNA regions) simultaneously, even from a single cell. In this note, we demonstrate the methods of this technique by using two different types of dinoflagellates, i.e. an armored freshwater species, Peridinium willei Huitfeld‐Kaas, and an unarmored marine species, Akashiwo sanguinea (Hirasaka) Hansen and Moestrup. By rotating the cell, photographs of all aspects of a single cell can be taken even using the SEM. The genes and DNA regions sequenced in these examples include a region of the ribosomal DNA (SSU, ITS1, 5.8S, ITS2, and part of the LSU) as well as part of the mitochondrial DNA‐encoded gene, cox1. This technique can be applied to both photosynthetic and heterotrophic dinoflagellates and will accelerate biodiversity studies.     

Morphology,ultrastructure and molecular phylogeny of cyst-producing Caladoa arcachonensis gen. et sp. nov. (Peridiniales,Dinophyceae) from France and Indonesia

The dinoflagellate order Peridiniales encompasses several well circumscribed families. However, the family level of some genera, such as Bysmatrum and Vulcanodinium, has remained elusive for many years. Four Peridinium-like strains were established from the Atlantic coast of France and North Sulawesi, Indonesia through cyst germination or isolation of single cells. The cyst-theca relationship was established on specimens from the French Atlantic. Their morphologies were examined using light, scanning and transmission electron microscopy. The cells were characterized by a much larger epitheca relative to the hypotheca, a large anterior sulcal (Sa) plate deeply intruding the epitheca and a small first anterior intercalary plate. The plate formula was identified as Po, cp, X, 4′, 3a, 7′′, 6C, 5S, 5′′′, 2′′′′, shared by Apocalathium, Chimonodinium, Fusiperidinium and Scrippsiella of the family Thoracosphaeraceae but the configuration of Sa plate and anterior intercalary plates is different. Transmission electron microscopy showed that the eyespot was located within a chloroplast comprising two rows of lipid globules and thus belongs to type A. All four strains were classified within a new genus Caladoa as C. arcachonensis gen. et sp. nov. Small subunit ribosomal DNA (SSU rDNA), partial large subunit ribosomal DNA (LSU rDNA) and internal transcribed spacer ribosomal DNA (ITS rDNA) sequences were obtained from all strains. Genetic distance based on ITS rDNA sequences between French and Indonesian strains reached 0.17, suggesting cryptic speciation in C. arcachonensis. The maximum likelihood and Bayesian inference analysis based on concatenated data from SSU and LSU rDNA sequences revealed that Caladoa is monophyletic and closest to Bysmatrum. Our results supported that Caladoa and Bysmatrum are members of the order Peridiniales but their family level remains to be determined. Our results also support that Vulcanodinium is closest to the family Peridiniaceae.     

ADAPTATION OF A PSYCHROPHILIC FRESHWATER DINOFLAGELLATE TO ULTRAVIOLET RADIATION1

Little is known about the UV photobiology of psychrophilic dinoflagellates, particularly in freshwater systems. We addressed the life strategies of Borghiella dodgei Moestrup, Gert. Hansen et Daugbjerg to cope with ambient levels of ultraviolet radiation (UVR) under cold conditions. Several physiological parameters related to growth, metabolism, and UVR protection were determined for 4 d in UVR‐exposed and control cells by applying stable isotope analysis, spectrophotometry, and liquid chromatography–mass spectrometry (LC/MS). In UVR‐exposed cells, assimilation of ¹⁵N and ¹³C and content of chl a and carotenoids, specifically diatoxanthin with respect to dinoxanthin and diadinoxanthin, were increased; furthermore, catalase activity showed a cyclic pattern with a strong increase after UVR exposure but a rapid return to preexposure levels. Both in UVR‐exposed and control cells, no lipid peroxidation of galactolipids was observed. However, in UVR‐exposed cells, content of galactolipids was higher and linked to an increase in monogalactosyldiacylglycerols (MGDGs). We concluded that Borghiella's adaptation to UVR depended on a general metabolic enhancement and efficient scavenging of oxygen radicals to mitigate and counteract damage. While Borghiella seemed to be well adapted to ambient UVR, the interactive effects of higher temperature and UVR on psychrophilic species in front of climate change merit further investigation.     

GYMNODINIUM COROLLARIUM SP. NOV. (DINOPHYCEAE)—A NEW COLD‐WATER DINOFLAGELLATE RESPONSIBLE FOR CYST SEDIMENTATION EVENTS IN THE BALTIC SEA1

A naked dinoflagellate with a unique arrangement of chloroplasts in the center of the cell was isolated from the northern Baltic proper during a spring dinoflagellate bloom (March 2005). Morphological, ultrastructural, and molecular analyses revealed this dinoflagellate to be undescribed and belonging to the genus Gymnodinium F. Stein. Gymnodinium corollarium A. M. Sundström, Kremp et Daugbjerg sp. nov. possesses features typical of Gymnodinium sensu stricto, such as nuclear chambers and an apical groove running in a counterclockwise direction around the apex. Phylogenetic analyses based on partial nuclear‐encoded LSU rDNA sequences place the species in close proximity to G. aureolum, but significant genetic distance, together with distinct morphological features, such as the position of chloroplasts, clearly justifies separation from this species. Temperature and salinity experiments revealed a preference of G. corollarium for low salinities and temperatures, confirming it to be a cold‐water species well adapted to the brackish water conditions in the Baltic Sea. At nitrogen‐deplete conditions, G. corollarium cultures produced small, slightly oval cysts resembling a previously unidentified cyst type commonly found in sediment trap samples collected from the northern and central open Baltic Sea. Based on LSU rDNA comparison, these cysts were assigned to G. corollarium. The cysts have been observed in many parts of the Baltic Sea, indicating the ecologic versatility of the species and its importance for the Baltic ecosystem.     

A further phylogenetic study of the heterotrophic dinoflagellate genus, Protoperidinium (Dinophyceae) based on small and large subunit ribosomal RNA gene sequences

The heterotrophic marine dinoflagellate genus Protoperidinium is the largest genus in the Dinophyceae. Previously, we reported on the intrageneric and intergeneric phylogenetic relationships of 10 species of Protoperidinium, from four sections, based on small subunit (SSU) rDNA sequences. The present paper reports on the impact of data from an additional 5 species and, therefore, an additional two sections, using the SSU rDNA data, but now also incorporating sequence data from the large subunit (LSU) rDNA. These sequences, in isolation and in combination, were used to reconstruct the evolutionary history of the genus. The LSU rDNA trees support a monophyletic genus, but the phylogenetic position within the Dinophyceae remains ambiguous. The SSU, LSU and SSU + LSU rDNA phylogenies support monophyly in the sections Avellana, Divergentia, Oceanica and Protoperidinium, but the section Conica is paraphyletic. Therefore, the concept of discrete taxonomic sections based on the shape of 1′ plate and 2a plate is upheld by molecular phylogeny. Furthermore, the section Oceanica is indicated as having an early divergence from other groups within the genus. The sections Avellana and Excentrica and a clade combining the sections Divergentia/Protoperidinium derived from Conica‐type dinoflagellates independently. Analysis of the LSU rDNA data resulted in the same phylogeny as that obtained using SSU rDNA data and, with increased taxon sampling, including members of new sections, a clearer idea of the evolution of morphological features within the genus Protoperidinium was obtained. Intraspecific variation was found in Protoperidinium conicum (Gran) Balech, Protoperidinium excentricum (Paulsen) Balech and Protoperidinium pellucidum Bergh based on SSU rDNA data and also in Protoperidinium claudicans (Paulsen) Balech, P. conicum and Protoperidinium denticulatum (Gran et Braarud) Balech based on LSU rDNA sequences. The common occurrence of base pair substitutions in P. conicum is indicative of the presence of cryptic species.     

Cryptic Speciation in the Genus Cryptoglena (Euglenaceae) Revealed by Nuclear and Plastid SSU and LSU rRNA Gene

The photosynthetic euglenoid genus Cryptoglena is differentiated from other euglenoid genera by having a longitudinal sulcus, one chloroplast, two large trough‐shaped paramylon plates positioned between the chloroplast and pellicle, and lack of metaboly. The genus contains only two species. To understand genetic diversity and taxonomy of Cryptoglena species, we analyzed molecular and morphological data from 25 strains. A combined data set of nuclear SSU and LSU and plastid SSU and LSU rRNA genes was analyzed using Bayesian, maximum likelihood, maximum parsimony, and distance (neighbor joining) methods. Although morphological data of all strains showed no significant species‐specific pattern, molecular data segregated the taxa into five clades, two of which represented previously known species: C. skujae and C. pigra, and three of which were designated as the new species, C. soropigra, C. similis, and C. longisulca. Each species had unique molecular signatures that could be found in the plastid SSU rRNA Helix P23_1 and LSU rRNA H2 domain. The genetic similarity of intraspecies based on nr SSU rDNA ranged from 97.8% to 100% and interspecies ranged from 95.3% to 98.9%. Therefore, we propose three new species based on specific molecular signatures and gene divergence of the nr SSU rDNA sequences.     

Laciniporus arabicus gen. et sp. nov. (Dinophyceae,Peridiniales), a new thecate,marine, sand‐dwelling dinoflagellate from the northern Indian Ocean (Arabian Sea)1

A new thecate, photosynthetic, sand‐dwelling marine dinoflagellate, Laciniporus arabicus gen. et sp. nov., is described from the subtidal sediments of the Omani coast in the Arabian Sea, northern Indian Ocean, based on detailed morphological and molecular data. Cells of L. arabicus are small (16.2–30.1 μm long and 13.1–23.2 μm wide), dorsoventrally compressed, with a small apical flap‐shaped projection pointing to the left. The thecal plate pattern is distinguished by minute first precingular plate and sulcus, which extends into the epitheca, with large anterior and right sulcal plates. The Kofoidian thecal tabulation is Po, X, 4′, 2a, 7′′, 6c, 6s, 5′′′, 2′′′′. Morphologically, the revealed plate pattern has an affinity to the Peridiniales, and LSU rDNA based phylogenetic analyses placed L. arabicus within the Thoracosphaeraceae, close to calcareous‐cyst producing scrippsielloids, predatory pfiesteriaceans, and photosynthetic freshwater peridinioids Chimonodinium lomnickii and Apocalathium spp. However, the thecal plate arrangement of L. arabicus differs noticeably from any currently described dinoflagellates, and the species stands out from closely related taxa by extensive differences in physiology and ecology.     

THE CALCAREOUS RESTING CYST OF PENTAPHARSODINIUM TYRRHENICUM COMB. NOV. (DINOPHYCEAE)1

While investigating dinoflagellate cyst assemblages in surface sediments of the Gulfs of Naples and Salerno (Mediterranean Sea), we found a new calcareous resting cyst. This cyst has a round to oval body surrounded by a thick mineral layer, which gives it the shape of a Napoleon hat, with a flat, oval face bearing the archeopyle and a convex keel on the opposite side. The cyst shape is variable in both natural samples and clonal cultures. The organic membrane underlying the calcareous covering is resistant to acetolysis, thus demonstrating the presence of sporopolleninlike material. The cyst germinated into a motile stage having the same morphological features and thecal plate pattern as Peridinium tyrrhenicum Balech. We believe the validity of the genus Pentapharsodinium Indelicato & Loeblich should be accepted. Based on the comparative examination of the species we collected and of a similar species, Pentapharsodinium trachodium Indelicato & Loeblich, we propose Pentapharsodinium tyrrhenicum as a new combination for Peridinium tyrrhenicum. The genus Pentapharsodinium also includes P. dalei Indelicato & Loeblich (= Peridinium faeroense Dale), which produces spiny, organic-walled cysts. The presence of species forming calcareous cysts and species producing noncalcareous cysts in the same genus raises questions about maintaining the family Calciodinellaceae. This family should only include calcareous cyst-forming peridinioids, in order to maintain a unified system of classification of fossil and recent dinoflagellates.     

Halosarpheia japonica sp. nov. (Halosphaeriales, Ascomycota) from marine habitats in Japan

A new Halosarpheia species, collected from driftwood from Hakkeijima beach, Yokohama, Japan, is described and illustrated and is compared with other species of the genus. The new fungus was growing together with its anamorph on a piece of decaying wood. SSU and LSU rDNA sequences for both morphs were 99% similar. Phylogenetic analyses of SSU and LSU rDNA sequences of the both morphs confirm their anamorph–teleomorph relationship and placed the new fungus in the Halosarpheia sensu stricto clade with high statistical support. Halosarpheia japonica is characterized by its polar appendage that is initially enclosed in a cellular sheath and dissolves in water, the appendage then swells to form a huge tree-like structure. The other three species currently included in Halosarpheia sensu stricto differ from H. japonica by having two polar appendages that uncoil in water to form long filaments.     

COMPARATIVE MORPHOLOGY AND MOLECULAR PHYLOGENETIC ANALYSIS OF THREE NEW SPECIES OF THE GENUS KARENIA (DINOPHYCEAE) FROM NEW ZEALAND1

Three new dinoflagellate species, Karenia papilionacea sp. nov., Karenia selliformis sp. nov., and Karenia bidigitata sp. nov., were compared with the toxic species Karenia mikimotoi (Miyake & Kominami ex Oda) G. Hansen & Moestrup, Karenia brevis (Davis) G. Hansen & Moestrup, and Karenia brevisulcata (Chang) G. Hansen & Moestrup using the same fixative. Distinguishing morphological characters for the genus Karenia included a smooth theca and a linear apical groove. The new species can be distinguished on the basis of morphological characters of vegetative cells that include the location and shape of the nucleus; the relative excavation of the hypotheca; the characteristics of apical and sulcal groove extensions on the epitheca; the cellular shape, size, and symmetry; the degree of dorsoventral compression; and the presence of an apical protrusion or carina. Species with pronounced dorsoventral compression swim in a distinctive fluttering motion. An intercingular tubular structure traversing the proximal and distal ends of the cingulum is common to the species of Karenia, Karlodinium micrum (Leadbeater & Dodge) J. Larsen, Gymnodinium pulchellum J. Larsen, and Gyrodinium corsicum Paulmier. Molecular phylogenetic analyses of rDNA sequence alignments show that the new species are phylogenetically distinct but closely related to K. mikimotoi and K. brevis.     

New taxa refresh the phylogeny and classification of pleurostomatid ciliates (Ciliophora,Litostomatea)

A high diversity of pleurostomatid ciliates has been discovered in the last decade, and their systematics needs to be improved in the light of new findings concerning their morphology and molecular phylogeny. In this work, a new genus, Protolitonotus gen. n., and two new species, Protolitonotus magnus sp. n. and Protolitonotus longus sp. n., were studied. Furthermore, 19 novel nucleotide sequences of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2 were collected to determine the phylogenetic relationships and systematic positions of the pleurostomatid ciliates in this study. Based on both molecular and morphological data, the results demonstrated that: (i) as disclosed by the sequence analysis of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2, Protolitonotus gen. n. is sister to all other pleurostomatids and thus represents an independent lineage and a separate family, Protolitonotidae fam. n., which is defined by the presence of a semi‐suture formed by the right somatic kineties near the dorsal margin of the body; (ii) the families Litonotidae and Kentrophyllidae are both monophyletic based on both SSU rDNA and LSU rDNA sequences, whereas Amphileptidae are non‐monophyletic in trees inferred from SSU rDNA sequences; and (iii) the genera Loxophyllum and Kentrophyllum are both monophyletic, whereas Litonotus is non‐monophyletic based on SSU rDNA analyses. ITS1‐5.8S‐ITS2 sequence data were used for the phylogenetic analyses of pleurostomatids for the first time; however, species relationships were less well resolved than in the SSU rDNA and LSU rDNA trees. In addition, a major revision to the classification of the order Pleurostomatida is suggested and a key to its families and genera is provided.     

Ultrastructure and LSU rDNA-based phylogeny of Peridinium lomnickii and description of Chimonodinium gen. nov. (Dinophyceae)

Several populations of Peridinium lomnickii were examined by SEM and serial section TEM. Comparison with typical Peridinium, Peridiniopsis, Palatinus and Scrippsiella species revealed significant structural differences, congruent with phylogenetic hypotheses derived from partial LSU rDNA sequences. Chimonodinium gen. nov. is described as a new genus of peridinioids, characterized by the Kofoidian plate formula Po, cp, x, 4', 3a, 7', 6c, 5s, 5', 2', the absence of pyrenoids, the presence of a microtubular basket with four or five overlapping rows of microtubules associated with a small peduncle, a pusular system with well-defined pusular tubes connected to the flagellar canals, and the production of non-calcareous cysts. Serial section examination of Scrippsiella trochoidea, here taken to represent typical Scrippsiella characters, revealed no peduncle and no associated microtubular strands. The molecular phylogeny placed C. lomnickii comb. nov. as a sister group to a clade composed of Thoracosphaera and the pfiesteriaceans. Whereas the lack of information on fine structure of the swimming stage of Thoracosphaera leaves its affinities unexplained, C. lomnickii shares with the pfiesteriaceans the presence of a microtubular basket and the unusual connection between two plates on the left side of the sulcus, involving extra-cytoplasmic fibres.     

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

The 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.     

A SURVEY OF THE STEROL COMPOSITION OF THE MARINE DINOFLAGELLATES KARENIA BREVIS,KARENIA MIKIMOTOI,AND KARLODINIUM MICRUM: DISTRIBUTION OF STEROLS WITHIN OTHER MEMBERS OF THE CLASS DINOPHYCEAE1

The sterol composition of different marine microalgae has been examined to determine the utility of sterols as biomarkers to distinguish members of various algal classes. For example, members of the class Dinophyceae possess certain 4‐methyl sterols, such as dinosterol, which are rarely found in other classes of algae. The ability to use sterol biomarkers to distinguish certain dinoflagellates such as the toxic species Karenia brevis Hansen and Moestrup, responsible for red tide events in the Gulf of Mexico, from other species within the same class would be of considerable scientific and economic value. Karenia brevis has been shown by others to possess two major sterols, (24S)‐4α‐methyl‐5α‐ergosta‐8(14),22‐dien‐3β‐ol (ED) and its 27‐nor derivative (NED), having novel structures not previously known to be present in other dinoflagellates. This prompted the present study of the sterol signatures of more than 40 dinoflagellates. In this survey, sterols with the properties of ED and NED were found in cultures of K. brevis and shown also to be the principal sterols of Karenia mikimotoi Hansen and Moestrup and Karlodinium micrum Larsen, two dinoflagellates closely related to K. brevis. They are also found as minor components of the more complex sterol profiles of other members of the Gymnodinium/Peridinium/Prorocentrum (GPP) taxonomic group. The distribution of these sterols is consistent with the known close relationship between K. brevis, K. mikimotoi, and K. micrum and serves to limit the use of these sterols as lipid biomarkers to a few related species of dinoflagellates.     

Morphology and Molecular Phylogeny of a New Marine,Sand-dwelling Dinoflagellate Genus,Pachena (Dinophyceae), with Descriptions of Three New Species

Marine benthic dinoflagellates are interesting not only because some epiphytic genera can cause harmful algal blooms but also for understanding dinoflagellate evolution and diversification. Our understanding of their biodiversity is far from complete, and many thecate genera have unusual tabulation patterns that are difficult to relate to the diverse known phytoplankton taxa. A new sand-dwelling genus, Pachena gen. nov., is described based on morphological and DNA sequence data. Three species were discovered in distant locations and are circumscribed, namely, P. leibnizii sp. nov. from Canada, P. abriliae sp. nov. from Spain, and P. meriddae sp. nov. from Italy. All species are tiny (about 9–23 μm long) and heterotrophic. Species are characterized by their tabulation (APC 4′ 3a 6′′ 5c 5s 5′′′ 2′′′′), an apical hook covering the apical pore, an ascending cingulum, and a sulcus with central list. The first anterior intercalary plate is uniquely “sandwiched” between two plates. The species share these features and differ in the relative sizes and arrangements of their plates, especially on the epitheca. The ornamentation of thecal plates is species-specific. The new molecular phylogenies based on SSU and LSU rDNA sequences contribute to understanding the evolution of the planktonic relatives of Pachena, the Thoracosphaeraceae.     

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.     

Biecheleria tirezensis sp. nov. (Dinophyceae,Suessiales), a new halotolerant dinoflagellate species isolated from the athalassohaline Tirez natural pond in Spain

A new euryhaline and eurythermal dinoflagellate species, Biecheleria tirezensis sp. nov., is described based on samples taken from an extreme environment, the athalassohaline and particularly sulphate-rich Tirez natural pond (Spain). This species is able to survive in salinities from almost fresh water up to 56 and over a 5–25°C temperature range. Thus, the ecological characteristics of this isolate differentiate it from other species of the same genus. Its morphology, as examined by light, scanning and transmission electron microscopy, shows that the cells are almost spherical, with several series of amphiesmal vesicles. It also has a single elongate amphiesmal vesicle (EAV) observed by SEM and the eyespot has a type E structure, typical of Biecheleria. Spherical and smooth cysts were observed in old cultures. The pigment composition is typical of a peridinin-containing dinoflagellate. Phylogeny inferred from nuclear rDNA SSU, ITS and LSU sequences showed the isolate belongs to the genus Biecheleria, closest to B. cincta and B. brevisulcata. Modelling and analysis of the secondary structure of its ITS2 region, and that of other species of the same genus and some representatives of the most closely related genera, indicated that the isolate represents a new species clearly separated from but related to B. cincta. The criterion of the presence of Compensatory Base Changes (CBCs) in the secondary structure of the ITS2 region as an indicator of species differentiation confirmed this, supporting the establishment of the Tirez pond isolate as a new species of Biecheleria.     

ON THE IDENTITY OF KARLODINIUM VENEFICUM AND DESCRIPTION OF KARLODINIUM ARMIGER SP. NOV. (DINOPHYCEAE), BASED ON LIGHT AND ELECTRON MICROSCOPY,NUCLEAR‐ENCODED LSU RDNA,AND PIGMENT COMPOSITION1

An undescribed species of the dinoflagellate genus Karlodinium J. Larsen (viz. K. armiger sp. nov.) is described from Alfacs Bay (Spain), using light and electron microscopy, pigment composition, and partial large subunit (LSU) rDNA sequence. The new species differs from the type species of Karlodinium (K. micrum (Leadbeater et Dodge) J. Larsen) by lacking rows of amphiesmal plugs, a feature presently considered to be a characteristic of Karlodinium. In K. armiger, an outer membrane is underlain by a complex system of cisternae and vacuoles. The pigment profile of K. armiger revealed the presence of chlorophylls a and c, with fucoxanthin as the major carotenoid. Phylogenetic analysis confirmed K. armiger to be related to other species of Karlodinium; thus forming a monophyletic genus, which, in the LSU tree, occupies a sister group position to Takayama de Salas, Bolch, Botes et Hallegraeff. The culture used by Ballantine to describe Gymnodinium veneficum Ballantine (Plymouth 103) was examined by light and electron microscopy and by partial LSU rDNA. Ultrastructurally, it proved identical to K. micrum (cultures Plymouth 207 and K. Tangen KT‐77D, the latter also known as K‐0522), and in LSU sequence, differed in only 0.3% of 1438 bp. We consider the two taxa to belong to the same species. This necessitates a change of name for the most widely found species, K. micrum, to K. veneficum. The three genera Karlodinium, Takayama, and Karenia constitute a separate evolutionary lineage, for which the new family Kareniaceae fam. nov. is suggested.