Cyst-theca Relationship Research Articles

New investigation of the cyst–motile relationship for Votadinium spinosum reveals a Protoperidinium claudicans species complex (Dinophyceae, Peridiniales)

Protoperidinium claudicans is a planktonic, heterotrophic, bioluminescent dinoflagellate species commonly found in neritic waters. It has long been considered to display phenotypic variation in its second anterior intercalary plate, which could vary between quadra, penta and hexa. The equivalent spinose, cordate cyst goes under the name of Votadinium spinosum. Here we perform incubation experiments with cysts from France, Canada, China and Japan, which demonstrate that P. claudicans forms a species complex, with at least two ribotypes with a penta configuration (P. claudicans) and one with a quadra configuration (P. carriae sp. nov.). A fossil-based cyst, V. multispinosum sp. nov., is described as the equivalent of P. carriae. Molecular phylogenetics using large subunit ribosomal DNA supports these observations. The cyst–theca relationships for Votadinium psilodora and another, undescribed, cordate, spineless Votadinium species are also reported from China. Macromolecular analyses of the cyst wall of V. multispinosum reveal it is comprised of a protein-rich carbohydrate compound. We show that this compound is not uncommon in dinoflagellate and ciliate cysts and that it is unlikely to preserve very well upon sedimentation and burial.

A new dinoflagellate Gonyaulax pospelovana with resting cysts resembling Spiniferites delicatus and its biogeography and ecology revealed by DNA metabarcoding

ABSTRACT Extant species of the dinoflagellate genus Gonyaulax are capable of producing resting cysts morphologically similar to different cyst-based genera, and their cyst-theca relationships are far from resolved. Here we have carried out germination experiments on several living cysts that resemble Spiniferites delicatus from the subtropical regions of China and Mexico. Both cyst and theca morphology were examined by light and scanning electron microscopy. A new species, Gonyaulax pospelovana, is described, characterized by a cingulum displacement and an overhang of twice its width, and two short antapical spines. The cysts of G. pospelovana had a granular surface and gonal processes with petaloid tips. Maximum likelihood and Bayesian inference analyses based on LSU and SSU rRNA gene sequences revealed that strains identified as G. pospelovana were monophyletic, forming a sister clade to Gonyaulax ellegaardiae and several presumable strains of Gonyaulax spinifera. One Chinese strain and two Mexican strains of G. pospelovana were examined for yessotoxin production using LC-MS/MS, but were not found to produce a detectable amount of toxins. Metabarcoding targeting the 18S V4 rRNA gene was performed on monthly collected samples in Xiamen Bay, China. A ZOTU (zero-radius operational taxonomic units) was detected that was consistent with G. pospelovana. Its maximum abundance was recorded in summer. Additionally, an OTU was identified as G. pospelovana from the Tara Oceans metabarcoding data, which occurred in the Indian and Pacific oceans at temperatures ranging from 28°C to 31°C, suggesting that it is a warm water species.

Cyst-theca relationships of Spiniferites bentorii, S. hyperacanthus, S. ramosus, S. scabratus and molecular phylogenetics of Spiniferites and Tectatodinium (Gonyaulacales, Dinophyceae)

ABSTRACT It is well known that modern resting cysts with morphologies matching those of species of the fossil genus Spiniferites germinate into motile cells of the genus Gonyaulax. Different Spiniferites species have been connected to a single Gonyaulax species, raising the question of whether they are over-classified. Through germination experiments of cysts with the morphological features of four species of Spiniferites, viz. S. bentorii, S. hyperacanthus, S. ramosus and S. scabratus, we established cyst-theca relationships. Cysts with the morphology of S. bentorii gave rise to vegetative, motile cells of Gonyaulax nezaniae sp. nov., which is characterized by two stout antapical spines. Cysts with S. hyperacanthus and S. ramosus morphologies germinated into Gonyaulax whaseongensis and G. spinifera, respectively. Cysts with S. scabratus morphology lacked a ventral pore and were attributed to Gonyaulax cf. spinifera. Gene sequences for SSU, LSU and/or ITS-5.8S rRNA were obtained from these four species, and from cysts with the morphology of Spiniferites belerius, S. mirabilis, S. lazus, Spiniferites cf. bentorii and Tectatodinium pellitum. The maximum likelihood and Bayesian inference analyses based on LSU and SSU rRNA gene sequences revealed that cysts assignable to Spiniferites formed a polyphyletic group, intermingled with Tectatodinium, Bitectatodinium, Ataxiodinium and Impagidinium, whereas Gonyaulax species appeared as monophyletic. From our results we inferred the phylogenetic positions of S. bentorii, S. mirabilis, S. lazus, S. scabratus, Tectatodinium pellitum and Gonyaulax digitale for the first time, supporting the idea that Spiniferites species are not over-classified and each of them may correspond to different Gonyaulax species.

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

ABSTRACTThe 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.

Morpho-molecular diversity and phylogeny of Bysmatrum (Dinophyceae) from the South China Sea and France

ABSTRACTThe dinoflagellate genus Bysmatrum encompasses five epibenthic or tide-pool species and has been characterized by separated anterior intercalary plates. In the present study, we obtained six strains of Bysmatrum from the South China Sea and French Atlantic coast by isolating single cells/cysts from plankton and sediment samples. All strains were examined with light microscopy and scanning electron microscopy. Based on morphological observations, three strains were identified as Bysmatrum subsalsum, characterized by the elongated and rectangular first and a hexagonal second anterior intercalary plate. They differ from each other in the number of sulcal lists and the configuration of the first anterior intercalary plate. One strain was identified as Bysmatrum gregarium and the other two as Bysmatrum granulosum. The cyst-theca relationship of B. subsalsum from the French Atlantic was established by incubation of the cyst, and the geochemical composition of the cyst wall was measured through micro-Fourier transform infrared spectroscopy. Bysmatrum subsalsum from Malaysia shows a bright red stigma in the sulcal area under light microscopy, which was confirmed with transmission electron microscopy: it was identified as a type B eyespot. Small subunit ribosomal DNA (SSU rDNA), partial large subunit ribosomal DNA (LSU rDNA) and internal transcribed spacer (ITS) sequences were obtained from all six strains. The maximum likelihood and Bayesian inference analysis based on concatenated SSU, ITS and LSU sequences revealed that Bysmatrum is monophyletic and nested within Peridiniales. Our strains of B. subsalsum form a new ribotype in the molecular phylogeny (designated as ribotype B). The genetic distance based on ITS sequences among Bysmatrum species ranged from 0.34 to 0.47 and those genetic distances at the intraspecific level of B. subsalsum could reach 0.41, supporting the possibility of hidden crypticity within B. subsalsum.

Cyst-Theca Relationship and Phylogenetic Position of Impagidinium caspienense Incubated from Caspian Sea Surface Sediments: Relation to Gonyaulax baltica and Evidence for Heterospory within Gonyaulacoid Dinoflagellates.

We investigate the cyst-theca relationship of Impagidinium caspienense. Through an incubation experiment, we succeeded in examining the motile stage. Additional molecular analysis of single-cyst PCR (LSU and SSU rDNA) reveal that the cyst is related to the species Gonyaulax baltica Ellegaard etal. (). The ability of this species to belong to two types of cyst-based genera (spiniferate and impagidinioid) suggests that environmental (particularly salinity) and not genetic factors explain the formation of both morphotypes by G.baltica, which provides evidence for heterospory in this species. The affiliation to G.baltica demonstrates that I.caspienense is not endemic to the Caspian Sea. The phylogenetic position of several other gonyaulacoid species is also documented: Impagidinium pallidum, Ataxiodinium choane, Pyxidinopsis psilata, Spiniferites belerius, and Spiniferites ramosus. The LSU and SSU rDNA based phylogenies suggest that the genera Impagidinium and Spiniferites are not monophyletic, and that P.psilata and A.choane are close to Gonyaulax verior and Gonyaulax polygramma, respectively. In addition, this study accentuates the importance of cyst morphology in the classification of the Gonyaulacales.

The cyst-theca relationship of the dinoflagellate cyst Trinovantedinium pallidifulvum, with erection of Protoperidinium lousianensis sp. nov. and their phylogenetic position within the Conica group

ABSTRACTWe establish the cyst-theca relationship of the dinoflagellate cyst species Trinovantedinium pallidifulvum Matsuoka 1987 based on germination experiments of specimens isolated from the Gulf of Mexico. We show that the motile stage is a new species, designated as Protoperidinium louisianensis. We also determine its phylogenetic position based on single-cell polymerase chain reaction (PCR) of a single cell germinated from the Gulf of Mexico cysts. To further refine the phylogeny, we determined the large subunit (LSU) sequence through single-cell PCR of the cyst Selenopemphix undulata isolated from Brentwood Bay (Saanich Inlet, BC, Canada). The phylogeny shows that P. louisianensis is closest to P. shanghaiense, the motile stage of T. applanatum, and is consistent with the monophyly of the genus Trinovantedinium. Selenopemphix undulata belongs to a different clade than Selenopemphix quanta (alleged cyst of P. conicum), suggesting that the genus Selenopemphix is polyphyletic. Trinovantedinium pallidifulvum is widely distributed with occurrences in the Gulf of Mexico, the North Atlantic, the northeast Pacific and southeast Asia. In addition, we illustrate the two other extant species, Trinovantedinium applanatum and Trinovantedinium variabile, and two morphotypes of Trinovantedinium. Geochemical analyses of the cyst wall of T. pallidifulvum indicate the presence of amide groups in agreement with other heterotrophic dinoflagellate species, although the cyst wall of T. pallidifulvum also includes some unique features.

The molecular characterization of the enigmatic dinoflagellate Kolkwitziella acuta reveals an affinity to the Excentrica section of the genus Protoperidinium

Kolkwitziella acuta is a cyst-forming dinoflagellate with a unique tabulation, occurring in freshwater to brackish environments of Eurasia and the USA. Based on the unique thecal plate arrangement, this species was previously interpreted as a missing link in the evolution between the genus Protoperidinium and the Diplopsalioideae. We isolated living K. acuta cysts from the south-western Caspian Sea and the central Baltic (Finland), and re-established the cyst-theca relationship. The cysts had a distinctive polar and dorsoventral compression and a characteristic wrinkly texture. The plate formula of the thecate stage was Po, X, 4′, 2a, 7′′, 3c+t, ?s, 5′′′, 1′′′′. Large subunit (LSU) and small subunit (SSU) ribosomal DNA sequences obtained through single-cell PCR of these cysts reveal that this species is close to Protoperidinum excentricum which belongs to the Excentrica section of the genus Protoperidinium. We suggest that it evolved from the genus Diplopsalis through a split of the apical and anterior intercalary plates. Furthermore, that the split of the antapical plates occurred several times independently in the evolution of Protoperidinium and Diplopsalioideae and is therefore a polyphyletic trait. The cyst of Kolkwitziella is considered an indicator of low salinities, specifically between 0 and 22 psu. Geochemical analysis of the cyst walls suggests a heterotrophic mode of nutrition.

Brigantedinium majusculum is the cyst of Protoperidinium sinuosum (Protoperidiniaceae, Dinophyceae)

:Two morphologically distinct species, Brigantedinium majusculum and Trinovantedinium applanatum, have been suggested as a cyst stage of Protoperidinium pentagonum; however, the cyst–theca relationships are still virtually undefined. In this study, we re-examined the motile stage of B. majusculum via thecal plate analysis together with a molecular phylogenetic analysis based on partial large-subunit ribosomal DNA gene sequences. The morphological features of motile cells germinated from B. majusculum were identical to those of Pr. sinuosum, and the molecular phylogenetic analysis showed that the germinated cells and B. majusculum are closely related to Pr. pentagonum and Pr. latissimum as a sister group. Additionally, high sequence divergences were observed between the germinated cells and B. majusculum as well as Pr. pentagonum and Pr. latissimum (18.0–19.5% according to P-values and 20.7–22.9% according to Kimura two-parameter values). On the basis of these results, our study indicates that B. majusculum should be accepted as the cyst of Pr. sinuosum.

Cyst–theca relationship and phylogenetic positions of the diplopsalioideans (Peridiniales, Dinophyceae), with description of Niea and Qia gen. nov.

:Nine genera have been assigned to the subfamily Diplopsalioideae, but since most have not been characterized molecularly and their cyst–theca relationships are largely unknown, the phylogenetic relationships among these genera are not well understood. Here we established the cyst–theca relationships of 11 species (Boreadinium breve, Diplopelta globula, Diplopsalis lenticula, Diplopsalopsis ovata, Lebouraia pusilla, Niea acanthocysta, Niea chinensis, Niea torta, Oblea rotunda, Preperidinium cf. meunieri and Qia lebouriae) belonging to nine genera by incubating cysts collected along the coast of China. In addition, we obtained 22 large-subunit ribosomal DNA sequences from the germinated motile cells of these 11 species by single-cell polymerase chain reaction. A new genus, Niea, was erected to encompass species with a plate formula identical to that of Oblea, that is, Po, X, 3′, 1a, 6′′, 3C+t, 5′′′, 2′′′′, but with an ortho 1′. Niea chinensis was described based on both theca and cyst morphology. The genus Oblea was emended to incorporate only species with a meta 1′. As a consequence, Oblea acanthocysta and O. torta were transferred to the newly erected genus Niea. A second new genus, Qia, was established to encompass Diplopsalis lebouriae, and the genus Diplopsalis was emended, differing from Qia in the shape of the anterior intercalary (1a) plate. Phylogenetic analyses were carried out using maximum likelihood and Bayesian inference. Molecular phylogeny revealed that the diplopsalioideans were not monophyletic and were subdivided into three clades. From our results, the shape of the first apical and anterior intercalary plates and the number of apical, anterior intercalary and antapical plates were useful characteristics to distinguish the diplopsalioideans at the genus level.

Cyst–theca relationship and phylogenetic positions of Protoperidinium (Peridiniales, Dinophyceae) species of the sections Conica and Tabulata, with description of Protoperidinium shanghaiense sp. nov.

:The genus Protoperidinium has been divided into several sections based on the shape of the first apical plate, the number/shape of anterior intercalary plates and the presence/absence of antapical horns/spines; however, phylogenetic relationships among these sections have not been fully explored. We examined the cyst–theca relationships of seven Protoperidinium species isolated from sediments along the Chinese coast. Ten partial nuclear-encoded large subunit ribosomal DNA (LSU rDNA) sequences were obtained by single-cell polymerase chain reaction (PCR) for seven species belonging to the Conica and Tabulata sections of the genus. New cyst–theca relationships were established for Protoperidinium biconicum and Protoperidinium humile, and the former was restricted to warmer waters. Brigantedinium majusculum was identified as the cyst of Protoperidinium pentagonum; whereas, Trinovantedinium applanatum corresponded to Protoperidinium shanghaiense sp. nov. Phylogenetic analyses based on LSU rDNA sequences were carried out using maximum likelihood and Bayesian inference and revealed that the theca-based section Conica was polyphyletic, and Tabulata was monophyletic. Surprisingly, some cyst-based genera (e.g. Brigantedinium, Selenopemphix) proved to be polyphyletic.

Cyst-theca relationship of the arctic dinoflagellate cyst Islandinium minutum (Dinophyceae) and phylogenetic position based on SSU rDNA and LSU rDNA.

Round brown spiny cysts constitute a morphological group common in high latitude dinoflagellate cyst assemblages. The dinoflagellate cyst Islandinium minutum (Harland et Reid) Head, Harland et Matthiessen is the main paleoecological indicator of seasonal sea-ice cover in the Arctic. Despite the importance of this cyst in paleoceanographical studies, its biological affinity has so far been unknown. The biological affinity of the species I.minutum and its phylogenetic position based on the small subunit ribosomal RNA gene (SSU rDNA) and the large subunit ribosomal RNA gene (LSU rDNA) were established from cyst incubation experiments in controlled conditions, optical and scanning electron microscopy, and single-cell PCR. The thecal motile cell obtained was undescribed. Although the motile cell was similar to Archaeperidinium minutum (Kofoid) Jörgensen, the motile cell of I.minutum lacked a transitional plate in the cingular series, which is present in Archaeperidinium spp. Islandinium minutum and Archaeperidinium spp. were paraphyletic in all phylogenetic analyses. Furthermore, Protoperidinium tricingulatum, which also lacks a transitional plate, was closely related to I.minutum and transfered to the genus Islandinium. Based on available data, it is clear that Islandinium is distinct from Archaeperidinium. Therefore, we considered Islandinium Head, Harland et Matthiessen as a non-fossil genus and emend its description, as well as the species I.minutum. This is the first description of a cyst-theca relationship and the first study that reports molecular data based on SSU rDNA and LSU rDNA on a species assigned to the genus Islandinium.

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 etal. 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 22-34psu.

Two new species formally attributed to Protoperidinium oblongum (Aurivillius) Park et Dodge (Peridiniales, Dinophyceae): Evidence from cyst incubation experiments

Cyst–theca relationships of the common armored dinoflagellate Protoperidinium oblongum were re-investigated by incubation experiments and molecular phylogenetic analysis. Living cysts provided for incubation experiments were collected from several places in Japan, including Omura Bay, West Japan and Lake Saroma in Hokkaido, North Japan. One hundred and four cysts were incubated and 18 motile cells were germinated from these cysts. To clarify their morphological characteristics both cysts and germinated thecate cells were observed, especially the archeopyle type, the number and shape of anterior intercalary plates, and the shape and development of apical and antapical horns. In order to provide molecular phylogenetic analysis, germinated cells from incubated cysts were examined for their LSU rDNA sequences. Results reveal that three morphologically different cysts produced three morphologically different thecate cells, which were previously known as Protoperidinium oblongum var. latidorsale, Protoperidinium oblongum var. inaequale, and Protoperidinium oblongum var. symmetricum. The molecular phylogenetic analysis demonstrated that the Oceania group in the genus Protoperidinium includes these three varieties as well as Protoperidinium divergens, Protoperidinium claudicans, and Protoperidinium steigingerae. This group is separated from Protoperidinium sensu stricto, and the three plankton forms are phylogenetically separate and independent species. Based on these facts, two new species, Protoperidinium quadrioblongum Sarai, Kawami et Matsuoka, the new name for Protoperidinium oblongum var. symmetricum and Protoperidinium paraoblongum Sarai, Kawami et Matsuoka for Protoperidinium oblongum var. inaequale are described.

Resting cysts of freshwater dinoflagellates in southeastern Georgian Bay (Lake Huron) as proxies of cultural eutrophication

Resting cysts attributed to the freshwater dinoflagellate genus Peridinium were found in surface sediments from Severn Sound, southeastern Georgian Bay (Lake Huron, Laurentian Great Lakes of North America). Two distinct cyst morphotypes were present and they were assigned to Peridinium wisconsinense Eddy, 1930 and Peridinium willei Huitfeldt-Kaas, 1900 by establishing cyst–theca relationships through germinations and single-cell LSU rDNA analysis on an excysted cell of Peridinium willei. Sediments recovered from deep, sheltered portions of Severn Sound and restricted basins like Honey Harbour contained between ~ 750 and 8500 cysts/cm 3. However, winnowing by bottom currents and high concentrations of dissolved oxygen adversely impact the dinoflagellate cyst record on the lakebed, and cyst concentrations in easily remobilized muds on bathymetric highs were < 100 cysts/cm 3. Down-core changes in the relative abundances of these two cyst morphotypes were attributed primarily to cultural eutrophication related to land-use changes around Severn Sound over the last six centuries. Cysts of Peridinium willei, a cosmopolitan dinoflagellate species that occurs in a broad range of temperature, pH and nutrient conditions, comprise 60–74% of the cysts identified in Ambrosia (ragweed)-rich sediments in the upper 20 cm of a gravity core taken from Honey Harbour. Euro-Canadian settlement and land-clearing that began in the Midland-Penetanguishene region around A.D. 1840 are evident in the increase in Ambrosia (ragweed), Gramineae (grasses) and other herbs (non-arboreal pollen) that mark the base of the Ambrosia zone (pollen zone 4) as well as an overall increase in terrigenous flux. In addition to siltation, this terrigenous flux increased the availability of limiting nutrients to the previously oligotrophic waters of Severn Sound, leading to increased cyst flux in Honey Harbour peaking at nearly 3000 cysts/cm 2/y in A.D. 1966, an order of magnitude higher than cyst fluxes prior to the Euro-Canadian Ambrosia zone. Peridinium wisconsinense was the more common dinoflagellate cyst species in Honey Harbour prior to Euro-Canadian settlement, when cyst flux was an order of magnitude lower. This is consistent with the restriction of this species to relatively warm, oligotrophic to mesotrophic lakes in North America. An earlier increase in P. willei at the expense of P. wisconsinense in the core from Honey Harbour within pollen zone 3 d (~ 700 to ~ 150 yBP) is attributed to earlier land-clearing by the Wendat (“Huron”), who practiced agriculture in the Penetanguishene peninsula between ~ A.D. 1450–1650. The cysts of these freshwater dinoflagellates thus appear to be sensitive to cultural eutrophication.

Protoperidinium minutum (Dinophyceae) from Portugal: cyst–theca relationship and phylogenetic position on the basis of single-cell SSU and LSU rDNA sequencing

Ribeiro S., Lundholm N., Amorim A. and Ellegaard M. 2010. Protoperidinium minutum (Dinophyceae) from Portugal: cyst–theca relationship and phylogenetic position on the basis of single-cell SSU and LSU rDNA sequencing. Phycologia 49: 48–63. DOI: 10.2216/09-11.1Round brown spiny cysts are common elements of Recent and Quaternary dinoflagellate cyst records and are often used to infer past climate conditions. Echinidinium and Islandinium, two cyst-based genera composed of round brown spiny cysts, are believed to have affinities within the Protoperidiniaceae. However, their biological counterparts are still virtually unknown. In this study, we examined the cyst–theca relationship of an Echinidinium-like cyst isolated from recent sediments of the Portuguese coast. The cysts (25–34 µm) had an intercalary theropylic archeopyle and numerous processes (4–9 µm) with tapered stems and minutely expanded tips. Germinated cells were identified as Protoperidinium minutum on the basis of theca morphology and tabulation. This taxon has a complicated taxonomic history and most likely represents a complex of species with very similar thecae but different cyst morphologies. To provide a first step in elucidating the phylogeny of P. minutum and its evolutionary relationship among the Protoperidiniaceae, we undertook the first molecular study of this taxon on the basis of small-subunit (SSU) and large-subunit (LSU) ribosomal (r)DNA genetic sequences obtained through single-cell polymerase chain reaction. On the basis of SSU rDNA analysis, P. minutum formed a clade together with the Diplopsaloideae, not grouping together with the other Protoperidinium species. LSU rDNA-based phylogenies indicate P. minutum as early divergent within the Protoperidiniaceae. The evolutionary significance of round brown spiny cysts produced by P. minutum-like species and diplopsalids is discussed.

Cyst-theca relationships of Gonyaulacales and Peridiniales (Dinophyceae) from Bahía Concepción, Gulf of California

Mediante ensayos de germinación se logró definir la relación quiste-teca de Gonyaulax spinifera (= Spiniferites mirabilis), Protoceratium globosum, Preperidinium meunieri (= Dubridinium caperatum), Protoperidinium denticulatum (= Brigantedinium irregulare) y Protoperidinium cf. thorianum. Los quistes se aislaron a partir del primer centímetro de sedimento superficial de muestras colectadas durante el periodo 2000-2002 en Bahía Concepción, Golfo de California. Los taxa identificados se ilustran con microfotografias, se describe la morfología de los estadios quístico y vegetativo, y también se incluye información sobre sinonimias, variación en talla y su distribución en México y en el mundo. Los ensayos de germinación permitieron describir por primera vez la relación quiste-teca de P. globosum, confirmar la presencia del estadio vegetativo de P. denticulatum en el Golfo de California y de la heterosporia en G. spinifera. También se establecieron con éxito cepas de G. spinifera y P. globosum.

Further examination of the cyst-theca relationship of Protoperidinium thulesense (Peridiniales, Dinophyceae) and the phylogenetic significance of round brown cysts

K. Matsuoka, H. Kawami, R. Fujii and M. Iwataki. 2006. Further examination of the cyst-theca relationship of Protoperidinium thulesense (Peridiniales, Dinophyceae) and the phylogenetic significance of round brown cysts. Phycologia 45: 632–641. DOI: 10.2216/05-42.1The heterotrophic armored dinoflagellate Protoperidinium thulesense has an unusual combination of morphological characters, i.e. the thecal plate arrangement of the motile cell resembles a typical Protoperidinium, whereas the shape and archeopyle of the cyst are like the diplopsalids. We have re-examined the cyst-motile relationship of P. thulesense by cyst incubation and thecal plate analysis together with a molecular phylogenetic study based on small subunit (SSU) rDNA sequences. Five isolates of P. thulesense, including motile cells and cysts, and three Protoperidinium and three diplopsalid species were examined by using the single cell PCR method. The thecal plate arrangement of the motile cells isolated from field samples and those germinated from cysts were identical. The plate formula was: Po, X, 3′, 3a, 7″, 3c+t, 4s, 5′″, 2″″. The cysts of P. thulesense were round and brown with a theropylic archeopyle, and rather similar to the diplopsalid species, Diplopsalis lenticula, D. lebourae, Gotoius abei and others. SSU rDNA sequence analysis reveals P. thulesense is closely related to the subgenus Protoperidinium, especially species of the section Conica, and distant from the diplopsalid species. The phylogenetic and taxonomic significances of Protoperidinium and diplopsalid round brown cysts with a theropylic archeopyle are discussed.

Morphological and LSU rDNA sequence variation within the Gonyaulax spinifera-Spiniferites group (Dinophyceae) and proposal of G. elongata comb. nov. and G. membranacea comb. nov.

Cultures were established from cysts of the cyst-based taxa Spiniferites elongatus and S. membranaceus. Motile cells and cysts from both cultures and sediment samples were examined using light and scanning electron microscopy. The cyst–theca relationship was established for S. elongatus. The motile cells have the tabulation pattern 2 pr, 4′, 6″, 6c, ≥ 4s, 6″′, 1p, 1″″, but they remain unattributable to previously described Gonyaulax species. There was large variation in process length and process morphology in cysts from both cultures and wild samples and there was variation in ornamentation and in the development of spines and flanges in motile cells. A new combination, G. elongata (Reid) Ellegaard et al. comb. nov. is proposed, following new rules of the International Code of Botanical Nomenclature that give genera based on extant forms priority over genera based on fossil forms. Extreme morphological variation in the cyst and motile stages of S. membranaceus is described and this species is also transferred to the genus Gonyaulax, as G. membranacea (Rossignol) Ellegaard et al. comb. nov. Approximately 1500 bp of large subunit (LSU) rDNA were determined for these two species and for G. baltica, G. cf. spinifera (= S. ramosus) and G. digitalis (= Bitectatodinium tepikiense). LSU rDNA showed sequence divergences similar to those estimated between species in other genera within the Gonyaulacales; a phylogeny for the Gonyaulacales was established, including novel LSU rONA sequences for Alexandrium margalefii, A. pseudogonyaulax and Pyrodinium bahamense var. compressum. Our results show that motile stages obtained from the germination of several cysts of the ‘fossil-based’ Spiniferites and B. tepikiense, which were previously attributed to ‘Gonyaulax spinifera group undifferentiated’, belong to distinct species of the genus Gonyaulax. These species show small morphological differences in the motile stage but relatively high sequence divergence. Moreover, this group of species is monophyletic, supported by bootstrap values of 100% in parsimony and maximum likelihood analyses.

NEW CALCAREOUS DINOFLAGELLATES (CALCIODINELLOIDEAE) FROM THE MEDITERRANEAN SEA1

Investigations on calcareous dinoflagellates from surface sediments from the Mediterranean Sea revealed 14 species, including one new genus and four previously undescribed species: Calciodinellum levantinum sp. nov., Calciodinellum elongatum nov. comb., Lebessphaera urania gen. nov. et sp. nov., and Scripp- siella triquetracapitata sp. nov. Furthermore, Fuettererella cf. tesserula, so far only known from the fossil record, was found. The cyst–theca relationships of C. levantinum and C. elongatum are given, based on strains established from water samples of the Mediterranean Sea and the Atlantic Ocean. This study gives an insight into the importance of the modern Mediterranean Sea as an unique region concerning calcareous cyst producing dinoflagellates.