Molecular phylogenetic study of the heterotrophic dinoflagellate genus Protoperidinium (Dinophyceae) inferred from small subunit rRNA gene sequences

In the present study, we investigated the intrageneric and intergeneric phylogenetic relationships of the heterotrophic marine dinoflagellate genus Protoperidinium. Using single‐cell polymerase chain reaction methods, we determined small subunit ribosomal RNA gene sequences for 10 Protoperidinium species belonging to four sections and two subgenera. Phylogenetic trees were constructed using maximum parsimony, neighbor joining and maximum likelihood methods. We found intraspecific variability of small subunit rDNA sequences in Protoperidinium conicum (Gran) Balech, Protoperidinium crassipes (Kofoid) Balech and Protoperidinium denticulatum (Gran et Braarud) Balech, but not in other species. The small subunit rDNA phylogeny revealed that the genus is monophyletic, but its phylogenetic position within the Dinophyceae could not be determined because of ambiguous basal topologies. Within the genus Protoperidinium, species of the subgenus Archaeperidinium with two anterior intercalary plates (2a) were shown to be monophyletic, but species of the subgenus Protoperidinium with three anterior intercalary plates (3a) were resolved as paraphyletic. The sections Avellana, Divergentia and Protoperidinium were shown to be monophyletic, while the section Conica was paraphyletic. Based on the trees obtained in the present study, most of the traditionally defined sections are supported by molecular phylogeny. It was also indicated that the section Avellana evolved from one of the Conica‐type dinoflagellates.     

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.     

New dinoflagellate species Protoperidinium haizhouense sp. nov. (Peridiniales,Dinophyceae), its cyst‐theca relationship and phylogenetic position within the Monovela group

The number of cingular plates has been used to differentiate Protoperidinium from Peridinium and related genera. Protoperidinium is characterized by the presence of three cingular plates plus a transitional plate (3C+t). However, many Protoperidinium species have been described that exhibit different cingular plate tabulations. How these species should be classified within the genus remains unclear. To address this question, the phylogenetic relationship of four Protoperidinium species, with three or four cingular plates and lacking a transitional plate, were examined in relationship to other Protoperidinium species. These four species were germinated from cysts deposited in surface sediments collected from the East China Sea, the Bohai Sea and the Yellow Sea. Three of the isolated species, P. tricingulatum, P. americanum and P. parthenopes, were described previously. The fourth is here described as P. haizhouense sp. nov. with the plate formula Po, X, 4′, 3a, 7′′, 3C, 6S, 5′′′, 2′′′′. Differences in the cyst stages of these four species, which can be taxonomically informative, were compared. Partial large subunit ribosomal DNA sequences were obtained by single‐cell polymerase chain reaction. Maximum‐likelihood and Bayesian inference showed that these four species, P. fukuyoi and Islandinium minutum form a monophyletic clade with maximal support. The genus as a whole, however, appeared polyphyletic. Our results suggest that the presence/absence of a transitional plate is significant in the phylogeny of Protoperidinium.     

Protoperidinium tricingulatum sp. nov. (Dinophyceae), a new motile form of a round,brown, and spiny dinoflagellate cyst

A small, broadly ovoidal and heterotrophic dinoflagellate containing round, brownish, and spiny cyst was found in the water column of Huibertsplaat in the Wadden Sea off the coast of the Netherlands. This dinoflagellate had these conspicuous morphological characters: a five‐sided first apical plate (1′), only three cingular plates, and an extremely small first antapical plate. Based on these morphological features, Protoperidinium tricingulatum Kawami, vanWezel, Koeman et Matsuoka is described as a new species. The flagellar pore of P. tricingulatum is covered with a small fin, which rises from the left side of the right sulcal plate to the large V‐shaped posterior sulcal plate. This feature suggests that P. tricingulatum is assigned to the Abé's Monovela Group. The cyst stage of P. tricingulatum was positively linked to the vegetative stage by comparison of the ribosomal 5.8S rDNA, internal transcribed spacers (ITS1 and ITS2). Living cysts of P. tricingulatum are round, brownish, and covered with many slender spines bearing capitate or cauliforate distal ends. The cyst also possesses a theropylic archeopyle formed by a slit corresponding to parasutures between three apical and two apical intercaraly plates. These morphological characters indicate that this species is morphologically related to two dinoflagellate cyst‐genera Islandinium and Echinidinium.     

MOLECULAR PHYLOGENY OF THE HETEROTROPHIC DINOFLAGELLATES,PROTOPERIDINIUM, DIPLOPSALIS AND PREPERIDINIUM (DINOPHYCEAE), INFERRED FROM LARGE SUBUNIT rDNA1

The genera Protoperidinium Bergh, Diplopsalis Bergh, and Preperidinium Mangin, comprised of species of marine, thecate, heterotrophic dinoflagellates in the family Protoperidinaceae Balech, have had a confused taxonomic history. To elucidate the validity of morphological groupings within the Protoperidinium and diplopsalids, and to determine the evolutionary relationships between these and other dinoflagellates, we undertook a study of molecular phylogeny using the D1–D3 domains of the large subunit (LSU) of the rDNA. Based on morphology, the 10 Protoperidinium species examined belonged to three subgenera and five morphological sections. Two diplopsalid species were also included. Single‐cell PCR, cloning, and sequencing revealed a high degree of intraindividual sequence variability in the LSU rDNA. The genus Protoperidinium appeared to be recently divergent in all phylogenetic analyses. In maximum parsimony and neighbor joining analyses, Protoperidinium formed a monophyletic group, evolving from diplopsalid dinoflagellates. In maximum likelihood and Bayesian analyses, however, Protoperidinium was polyphyletic, as the lenticular, diplopsalid heterotroph, Diplopsalis lenticula Bergh, was inserted within the Protoperidinium clade as basal to Protoperidinium excentricum (Paulsen) Balech, and Preperidinium meunieri (Pavillard) Elbrächter fell within a separate clade as a sister to the Oceanica and Protoperidinium steidingerae Balech. In all analyses, the Protoperidinium were divided into two major clades, with members in the Oceanica group and subgenus Testeria in one clade, and the Excentrica, Conica, Pellucida, Pyriforme and Divergens sections in the other clade. The LSU rDNA molecular phylogeny supported the historical morphologically determined sections, but not a simple morphology based model of evolution based on thecal plate shape.     

PROTOPERIDINIUM BELIZEANUM SP. NOV. (DINOPHYCEAE) FROM MANATEE CAY,BELIZE, CENTRAL AMERICA1

A new marine heterotrophic dinoflagellate species, Protoperidinium belizeanum sp. nov., from a coral reef‐mangrove pond was identified from scanning electron micrographs. Recognition of this new species was based on unique features of the thecal morphology, which included cell size and shape, presence of short and wide postcingular plates, sulcal architecture, antapical spines, and intricate thecal plate patterns of ridged hexagonal depressions. The thecal plate formula is as follows: Po, X, 4′, 3a, 7″, 4C (3+t), 6S, 5?, 2″″. Species association of P. be‐lizeanum sp. nov. within the genus Protoperidinium, its habitat, and associated dinoflagellates species are discussed.     

Abstracts of papers read at the Winter Meeting, 1961

Herdmania litoralis is a heterotrophic, sand-dwelling dinoflagellate with morphological characters that do not provide clear evidence for its systematic position in any existing family of dinoflagellates. Protoperidinium minutum is a heterotrophic, planktonic species that has a typical tabulation for the genus Protoperidinium. In order to infer the phylogenetic positions of these two species more confidently, we characterized the thecal plate patterns and determined small-subunit and large-subunit ribosomal DNA sequences (SSU rDNA and LSU rDNA, respectively) from both species. Intraindividual and intraspecific diversity of SSU and LSU rDNA data were characterized in H. litoralis using a combination of single-cell PCR approaches and analyses of PCR clones derived from multi-cell DNA extractions. The results of the molecular phylogenetic analyses demonstrated a novel, well-supported clade comprising both sand-dwelling species (H. litoralis and Thecadinium dragescoi) and planktonic species (P. minutum). Because the establishment of this clade also demonstrated that P. minutum is not a member of Protoperidinium, we reinstated and emended the genus Archaeperidinium Jörgensen 1912 Jörgensen, E. 1912. Bericht über die von der schwedischen hydrographisch-biologischen Kommission in den schwedischen Gewässern in den Jahren 1909–10 eingesammelten Planktonproben. Svenska Hydrograph.-Biol. Komm. Skr., 4: 1–20.  [Google Scholar].     

CULTURE OF THE HETEROTROPHIC DINOFLAGELLATE PROTOPERIDINIUM CRASSIPES (DINOPHYCEAE) WITH NONCELLULAR FOOD ITEMS1

The genus Protoperidinium is an assemblage of heterotrophic dinoflagellates, several species of which have been successfully cultured in the past using various photosynthetic algae as a food source. We succeeded in culturing Protoperidinium crassipes (Kof.) Balech on three separate occasions for periods ranging from 2 to 21 months using rice flour as a food source. In these cultures, unusual small types of cells that were never observed to actively feed sometimes appeared. We confirmed that P. crassipes in culture exhibited bioluminescence.     

Cyst‐theca relationship of a new dinoflagellate with a spiny round brown cyst,Protoperidinium lewisiae sp. nov., and its comparison to the cyst of Oblea acanthocysta

Round spiny brown cysts with apiculocavate processes were isolated from sediments of Lake Saroma, Japan, Changle Harbor, East China Sea, China, Jinzhou Harbor, Bohai Sea, China, and San Pedro Harbor, California, USA. Superficially similar round spiny brown cysts of the species, Oblea acanthocysta were, for comparison, restudied through light microscopy and scanning electron microscopy (SEM) and by sequencing of small subunit (SSU) and large subunit (LSU) rDNA obtained through a single cyst from Lake Saroma. These morphological measurements and SEM observations showed that the new cysts can be discriminated from O. acanthocysta by the archeopyle, number of processes, shape of process bases and its apiculocavate processes. Based on LSU sequences, the most closely related species was Protoperidinium monovelum, for which no cyst stage has been described so far. However, the thecal morphology of the specimens found in this study differed from P. monovelum in details of the sulcal plates and shape of apical pore and 2a plate. We therefore describe Protoperidinium lewisiae sp. nov., which can be found in estuarine subtropical to temperate waters of the Pacific Ocean.     

DESCRIPTION OF A NEW GENUS OF PFIESTERIA‐LIKE DINOFLAGELLATE,LUCIELLA GEN. NOV. (DINOPHYCEAE), INCLUDING TWO NEW SPECIES: LUCIELLA MASANENSIS SP. NOV. AND LUCIELLA ATLANTIS SP. NOV.1

A new genus of Pfiesteria‐like heterotrophic dinoflagellate, Luciella gen. nov., and two new species, Luciella masanensis sp. nov. and Luciella atlantis sp. nov., are described. These species commonly occur with other small (<20 μm) heterotrophic and mixotrophic dinoflagellates in estuaries from Florida to Maryland and the southern coast of Korea, suggesting a possible global distribution. An SEM analysis indicates that members of the genus Luciella have the enhanced Kofoidian plate formula of Po, cp, X, 4′, 2a, 6″, 6c, PC, 5+s, 5?, 0p, and 2″″. The two four‐sided anterior intercalary plates are diamond shaped. The genus Luciella differs from the other genera in the Pfiesteriaceae by a least one plate in the plate tabulation and in the configuration of the two anterior intercalary plates. An SSU rDNA phylogenetic analysis confirmed the genus as monophyletic and distinct from the other genera in the Pfiesteriaceae. The morphology of Luciella masanensis closely resembles Pfiesteria piscicida Steid. et J. M. Burkh. and other Pfiesteria‐like dinoflagellates in size and shape, making it easily misidentified using LM. Luciella atlantis, in contrast, has a more distinctive morphology. It can be distinguished from L. masanensis and other Pfiesteria‐like organisms by a larger cell size, a more conical‐shaped epitheca and hypotheca, larger rhombic‐shaped intercalary plates, and an asymmetrical hypotheca. The genus Luciella is assigned to the order Peridiniales and the family Pfiesteriaceae based on plate tabulation, plate pattern, general morphology, and phylogenetic analysis.     

The genus Glochidinium gen. nov., with two species: G. penardiforme comb. nov. and G. platygaster sp. nov. (Peridiniaceae)

Glochidiniumgen. nov., a ncw genus of Peridiniaceae based on Peridinium penardiforme Lindemann, is herewith erected. Its plate formula is: Po+X+4′+6′′+3C+4S+5′′′+2′′′′ Main diagnostic characters of this new genus are the presence of only 3 cingular plates (it lacks the transitionalone), the third cingular contacting the anterior sulcal plate, and an unusual sulcus holding a small triangular posterior sulcal plate. The thecal morphology and structure of two freshwater planktic species of the genus are described on the basis of LM and SEM observations. G. penardiforme comb. nov. is an infrequent species, albeit widely distributed world-wide. It has been recorded under the names of Peridinium, Glenodinium and Peridiniopsis. Peculiar features in the tabulation of the furrows and of the surface sculpture show that the species does not fit any of the known genera, for which reason the new genus Glochidinium is established. G. platygaster sp. nov., the second species included in the genus, differs from the former by its elongated body, the regular pentagonal shape of its large first apical plate, an equally large sulcal anterior plate, and well developed sculpture, chiefly on the antapical plates. Glochidinium penardiforme and G. platygaster were found in some reservoirs from central and northern Argentina. G. penardiforme was also found in several Argentine rivers and ponds.     

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.     

SEASONAL VARIABILITY OF THE ORGANIC‐WALLED DINOFLAGELLATE CYST PRODUCTION IN THE COASTAL UPWELLING REGION OFF CAPE BLANC (MAURITANIA): A FIVE‐YEAR SURVEY1

A 5‐year sediment trap survey in the upwelling area off Cape Blanc (NW Africa) provides information on the seasonal and annual resting cyst production of dinoflagellates, their sinking characteristics and preservation potential. Strong annual variation in cyst production characterizes the region. Cyst production of generally all investigated species, including Alexandrium pseudogonyaulax (Biecheler) T. Horig. ex T. Kita et Fukuyo (cyst genus Impagidinium) and Gonyaulax spinifera (Clap. et J. Lachm.) Diesing (cyst genus Nematosphaeropsis) was enhanced with increasing upper water nutrient and trace‐element concentrations. Cyst production of Lingulodinium polyedrum (F. Stein) J. D. Dodge was the highest at the transition between upwelling and upwelling‐relaxation. Cyst production of Protoperidinium americanum (Gran et Braarud) Balech, Protoperidinium monospinum (Paulsen) K. A. F. Zonn. et B. Dale, and Protoperidinium stellatum (D. Wall) Balech, and heterotrophic dinoflagellates forming Brigantedinium spp. and Echinidinium aculeatum Zonn., increased most pronouncedly during upwelling episodes. Production of Protoperidinium conicum (Gran) Balech and Protoperidinium pentagonum (Gran) Balech cysts and total diatom valves were related, providing evidence of a predator–prey relationship. The export cyst‐flux of E. aculeatum, P. americanum, P. monospinum, and P. stellatum was strongly linked to the flux of total diatom valves and CaCO₃, whereas the export production of Echinidinium granulatum Zonn. and Protoperidinium subinerme (Paulsen) A. R. Loebl. correlated with total organic carbon, suggesting potential consumption of diatoms, prymnesiophytes, and organic matter, respectively. Sinking velocities were at least 274 m · d^?1, which is in range of the diatom‐ and coccolith‐based phytoplankton aggregates and “slower” fecal pellets. Species‐selective degradation did not occur in the water column, but on the ocean floor.     

A New Heterotrophic Dinoflagellate from the North‐eastern Pacific,Protoperidinium fukuyoi: Cyst–Theca Relationship,Phylogeny, Distribution and Ecology

The cyst–theca relationship of Protoperidinium fukuyoi n. sp. (Dinoflagellata, Protoperidiniaceae) is established by incubating resting cysts from estuarine sediments off southern Vancouver Island, British Columbia, Canada, and San Pedro Harbor, California, USA. The cysts have a brown‐coloured wall, and are characterized by a saphopylic archeopyle comprising three apical plates, the apical pore plate and canal plate; and acuminate processes typically arranged in linear clusters. We elucidate the phylogenetic relationship of P. fukuyoi through large and small subunit (LSU and SSU) rDNA sequences, and also report the SSU of the cyst‐defined species Islandinium minutum (Harland & Reid) Head et al. 2001. Molecular phylogenetic analysis by SSU rDNA shows that both species are closely related to Protoperidinium americanum (Gran & Braarud 1935) Balech 1974. Large subunit rDNA phylogeny also supports a close relationship between P. fukuyoi and P. americanum. Three subgroups in total are further characterized within the Monovela group. The cyst of P. fukuyoi shows a wide geographical range along the coastal tropical to temperate areas of the North‐east Pacific, its distribution reflecting optimal summer sea‐surface temperatures of ~14–18 °C and salinities of 22–34 psu.     

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.     

Studies on woloszynskioid dinoflagellates VI: description of Tovellia aveirensis sp. nov. (Dinophyceae), a new species of Tovelliaceae with spiny cysts

A new species of Tovellia, T. aveirensis, is described on the basis of light (LM) and scanning electron microscopy (SEM) of motile cells and resting cysts, complemented with transmission electron microscopy (TEM) of flagellate cells and phylogenetic analysis of partial sequences of the large subunit ribosomal rRNA gene. Both vegetative cells and several stages of a life cycle involving sexual reproduction and the production of resting cysts were examined in cultures established from a tank in the University of Aveiro campus. Vegetative cells were round and little compressed dorsoventrally; planozygotes were longer and had a proportionally larger epicone. Chloroplast lobes were shown by TEM to radiate from a central, branched pyrenoid, although this was difficult to ascertain in LM. The amphiesma of flagellate cells had mainly 5 or 6-sided vesicles with thin plates, arranged in 5–7 latitudinal series on the epicone, 3–5 on the hypocone. The cingulum had 2 rows of plates, the posterior row extending into the hypocone and crossed by a series of small projecting knobs along the lower edge of the cingulum. A line of narrow amphiesmal plates extended over the cell apex, from near the cingulum on the ventral side to the middle of the dorsal side of the epicone. Eight or 9 narrow amphiesmal plates lined each side of this apical line of plates (ALP). Resting cysts differed from any described before in having numerous long, tapering spines with branched tips distributed over most of the surface. Most mature cysts showed an equatorial constriction. Neither cysts nor motile cells were seen to accumulate red cytoplasmic bodies in any stage of the cultures. The phylogenetic analysis placed, with high statistical support, the new species within the genus Tovellia; it formed a clade, with moderate support, with T. sanguinea, a species notable for its reddening cells.