The significance of sexual versus asexual cyst formation in the life cycle of the noxious dinoflagellate Alexandrium peruvianum

Alexandrium peruvianum (Balech et Mendiola) is a noxious phototrophic marine dinoflagellate. During the life cycle of this species, two kinds of cysts are produced: resting cysts, which are long-lasting and double-walled, and temporary cysts, which are short-lasting and thin-walled. In addition, short-lasting, but resting-like cysts can also be formed. Although it is crucial to identify sexual events in a dinoflagellate population, sexual and asexual cysts are morphologically very similar in this species. Therefore, we studied the complete life cycle and the nature of the cyst-like stages formed after individual isolation of specimens and crossing of clonal cultures established from germination of wild resting cysts. Asexual division in A. peruvianum takes place either in the motile stage by sharing of the theca (desmoschisis), or inside a vegetative cyst (temporary cyst), from which two, or at times four or six naked daughter cells can originate. The daughter cells completely synthesize new cell walls (eleutheroschisis). Sexuality was confirmed by the presence of fusing gamete pairs and longitudinally biflagellated planozygotes after out-crossing of compatible clonal strains. However, the clonal cultures had low levels of self-compatibility, since a flow cytometry analysis showed that synchronized self-crosses produced few zygotes (<5%). After isolation of individual cells, it was proved that the fate of the planozygotes depended on the nutritional status of the isolation media. Most of the planozygotes isolated to replete medium (L1) divided, whereas in medium lacking nitrates (L-N) or phosphates (L-P) they formed temporary, thin-walled cysts. Temporary cysts formed in L1 were always uninucleated and gave rise to one cell, while those formed in L-N or L-P produced 1–6 small cells. In addition, resting cysts were formed in culture, but never after individual planozygote isolation. Resting cysts were uninucleated and needed maturation time before entering dormancy. The resting cysts were considered sexual products, since longitudinally biflagellate germlings were liberated after germination in all cases studied. Mature resting cysts (52.3 ± 3.0 μm) had a dormancy period of 1–3 months, whereas temporary asexual cysts (32.5 ± 5.4 μm) germinated in less than 7 days.     

THE SEXUAL LIFE CYCLES OF PFIESTERIA PISCICIDA AND CRYPTOPERIDINIOPSOIDS (DINOPHYCEAE)1

Sexual life cycle events in Pfiesteria piscicida and cryptoperidiniopsoid heterotrophic dinoflagellates were determined by following the development of isolated gamete pairs in single‐drop microcultures with cryptophyte prey. Under these conditions, the observed sequence of zygote formation, development, and postzygotic divisions was similar in these dinoflagellates. Fusion of motile gamete pairs each produced a rapidly swimming uninucleate planozygote with two longitudinal flagella. Planozygotes enlarged as they fed repeatedly on cryptophytes. In <12 h in most cases, each planozygote formed a transparent‐walled nonmotile cell (cyst) with a single nucleus. Zygotic cysts did not exhibit dormancy under these conditions. In each taxon, dramatic swirling chromosome movements (nuclear cyclosis) were found in zygote nuclei before division. In P. piscicida, nuclear cyclosis occurred in the zygotic cyst or apparently earlier in the planozygote. In the cryptoperidiniopsoids, nuclear cyclosis occurred inthe zygotic cyst. After nuclear cyclosis, a single cell division occurred in P. piscicida and cryptoperidiniopsoid zygotic cysts, producing two offspring that emerged as biflagellated cells. These two flagellated cells typically swam for hours and fed on cryptophytes before encysting. A single cell division in these cysts produced two biflagellated offspring that also fed before encysting for further reproduction. This sequence of zygote development and postzygotic divisions typically was completed within 24 h and was confirmed in examples from different isolates of each taxon. Some aspects of the P. piscicida sexual life cycle determined here differed from previous reports.     

THE LIFE HISTORY OF ALEXANDRIUM TAYLORI (DINOPHYCEAE)

The gonyaulacoid dinoflagellate Alexandrium taylori Balech is reported for the first time from Italian waters. In July 1997, nonmotile stages of this species, both temporary and sexual resting cysts, were found in surface Ionian coastal waters (Mediterranean Sea) producing localized brownish-yellow patches. Clonal cultures were established, and the life history of A. taylori was studied in the laboratory. Asexual reproduction took place during a motile phase and produced two daughter cells remaining temporarily attached in pairs. This species exhibited isogamy. Small gametes were produced from vegetative cells through the release of a division cyst and multiple fission of the protoplast. Isogametes from the same clonal strain fused and underwent sexual reproduction, forming planozygotes that subsequently developed storage bodies and dark pigmentation. The maturation of the planozygote into hypnozygote also involved an increase in size and final shedding of flagella and theca. Hypnozygotes germinated within 15 days of their formation, and a naked planomeiocyte emerged from the archeopyle to undergo successive divisions and reestablish a haploid motile population.     

MULTIPLE ROUTES OF SEXUALITY IN ALEXANDRIUM TAYLORI (DINOPHYCEAE) IN CULTURE1

Alexandrium taylori Balech is a cyst‐forming dinoflagellate species responsible for recurrent blooms in Mediterranean coastal waters. The nuclear development of the cells during the sexual cycle and the effect of different external nitrate and phosphate levels were studied. Nuclear fusion of gametes occurred 6–12 h after the complete cytoplasmic fusion. The U‐shaped nuclei fused through the end of one nucleus and the mid‐area of the other. The mobile and biflagellated zygote had a large, U‐shaped nucleus and may follow three different fates: direct division, short‐term encystment (ecdysal), and long‐term encystment (resting). Ecdysal cysts may divide in >24–96 h into two, four, six, or eight cells before germinating. Meiosis presumably occurred in three locations: in the planozygote, within the ecdysal cyst, and in the planomeiocyte (germling) liberated either from ecdysal or resting cysts. The effects of nutrients on these routes were studied in individually isolated sexual stages. (1) Direct divisions occurred mainly under replete conditions (L1), whereas no direct planozygote divisions were recorded in media with no phosphate added (L‐P). (2) Short‐term encystment was larger in media lacking phosphate (L‐P and L/30) than in medium with no nitrate added (L‐N) or under replete conditions (L1). (3) Long‐term encystment was only observed in medium with no nitrate added (L‐N). The long‐lived resting cyst, not previously described for this species, had a clear double wall, an irregular shape, a flat morphology, and a middle orange spot. No cysts germinated in 1–2 months, whereas 86% of the cysts germinated 2–3 months after being formed. A flow cytometry analysis showed that sexual induction and zygote formation were very fast and highly common processes, zygotes being nearly half of the population at days 3 and 5 after the induction of sexuality in the cultures.     

Sexual resting cyst production by the dinoflagellate Akashiwo sanguinea: a potential mechanism contributing to the ubiquitous distribution of a harmful alga

The dinoflagellate Akashiwo sanguinea is a well known, cosmopolitan harmful microalga that frequently forms harmful algal blooms (HABs) in marine estuaries from temperate to tropical waters, and has posed a severe threat to fish, shellfish, and sea birds. Therefore, it is important to understand the ecology of this species, particularly the mechanisms regulating its ubiquitous geographic distribution and frequent recurrence of. To date, the mechanisms regulating distribution and recurrence of HABs by this species have been poorly understood. While resting cyst production can play a central role in the geographic expansion and initiation of HABs, studies of the life cycle of this alga, including cyst production, have been lacking. Here, we demonstrate that A. sanguinea produces sexual resting cysts homothallically. We present evidence for cell pairs in sexual mating, biflagellated planozygote formation, and cysts of different morphologies, and we describe time series for germination of cysts to germlings with two longitudinal flagella, along with studies of possible factors affecting cyst production. Phylogenetic analysis of large sub‐unit rDNA sequences revealed a monophyly of this species and thus possibly a recent common ancestor for all global populations. The discovery of resting cyst production by A. sanguinea suggests its frequent recurrence of blooms and global distribution may have been facilitated by the natural and anthropogenic transport of resting cysts.     

LIFE CYLE AND SEXUALITY OF THE FRESHWATER RAPHIDOPHYTE GONYOSTOMUM SEMEN (RAPHIDOPHYCEAE)1

Previously unknown aspects in the life cycle of the freshwater flagellate Gonyostomum semen (Ehrenb.) (Raphidophyceae) are described here. This species forms intense blooms in many northern temperate lakes, and has increased in abundance and frequency in northern Europe during the past decades. The proposed life cycle is based on observations of life cycle stages and transitions in cultures. Viable stages of the life cycle were individually isolated and monitored by time‐lapse photography. The most common processes undertaken by the isolated cells were: division, fusion followed by division, asexual cyst formation, and sexual cyst formation. Motile cells divided by two different processes. One lasted between 6 and 24 h and formed two cells with vegetative cell size and with or without the same shape. The second division process lasted between 10 and 20 min and formed two identical cells, half the size of the mother cell. Planozygotes formed by the fusion of hologametes subsequently underwent division into two cells. Asexual cyst‐like stages were spherical, devoid of a thick wall and red spot, and germinated in 24–48 h. Heterogamete pairs were isogamous, and formed an angle of 0–90° between each other. Planozygote and sexual cyst formation were identified within strains established from one vegetative cell. The identity of these strains, which was studied by an amplified fragment length polymorphism analysis, was correlated with the viability of the planozygote. Resting cyst germination was described using cysts collected in the field. The size and morphology of these cysts were comparable with those formed sexually in culture. The excystment rate was higher at 24°C than at 19 or 16°C, although the cell liberated during germination (germling) was only viable at 16°C. The placement of G. semen within the Raphidophyceae family was confirmed by sequence analysis of a segment of the 18S ribosomal DNA.     

NUCLEAR FEATURES AND EFFECT OF NUTRIENTS ON GYMNODINIUM CATENATUM (DINOPHYCEAE) SEXUAL STAGES1

Gymnodinium catenatum Graham is an unarmored, cyst‐forming dinoflagellate species responsible for outbreaks of paralytic shellfish poisoning. The nuclear development of the cells during the sexual cycle and the effect of different nitrate and phosphate external levels on sexual stages were studied. Nuclear fusion of gametes occurred before or at the same time as cytoplasmic fusion. During this process, either both nuclei migrated to a central area in the sulcal region, or only one of them migrated to the other nucleus. The motile and longitudinally biflagellated zygote presented a large, pear‐shaped nucleus, and either divided or encysted. Planozygotes and germlings underwent similar division processes, which suggested an uncoordinated meiosis in both encysting and non‐encysting zygotes. Encystment in culture was greater under low nitrate and phosphate limitation (L/15) than when only one or neither of these nutrients were added (L‐N, L‐P, and ‐N‐P). However, planozygotes individually monitored achieved the maximum encystment (40%) in a medium with no phosphate or nitrate added (‐N‐P), while most of them divided (70%–90%) in replete (L1) or half‐replete (L‐N and L‐P) media. Low levels of nitrate in the medium of cyst formation promoted a deficient development of the cyst wall. On the other hand, low phosphate levels in the medium of germination prevented both planozygote and germling division and lowered the final germination frequencies of cysts. The minimum dormancy, with an average value of 13.7±5.5 days, was not affected by any of the nutritional conditions studied.     

VEGETATIVE REPRODUCTION AND SEXUAL LIFE CYCLE OF THE TOXIC DINOFLAGELLATE GYMNODINIUM CATENATUM FROM TASMANIA,AUSTRALIA1

The toxic, chain-forming dinoflagellate Gymnodinium catenatum Graham was cultured from vegetative cells and benthic resting cysts isolated from estuarine waters in Tasmania, Australia. Rapidly dividing, log phase cultures formed long chains of up to 64 cells whereas stationary phase cultures were composed primarily of single cells (23-41 pm long, 27-36 pm wide). Vegetative growth (mean doubling time 3-4 days) was optimal at temperatures from 14.5-20° C, salinities of 23-34% and light irradiances of 50-300 μE·m^?2·s^?1. The sexual life cycle of G. catenatum was easily induced in a nutrient-deficient medium, provided compatible opposite mating types were combined (heterothallism). Gamete fusion produced a large (59-73 μm long, 50-59 μm wide) biconical, posteriorly biflagellate planozygote (double longitudinal flagellum) which after several days lost one longitudinal flagellum and gradually became subspherical in shape. This older planozygote stage persisted for up to two weeks before encysting into a round, brown resting cyst (42-52 μm diam; hypnozygote) with microreticulate surface ornamentation. Resting cysts germinated after a dormancy period as short as two weeks under our culture conditions, resulting in a single, posteriorly biflagellate germling cell (planomeiocyte). This divided to form a chain of two cells, which subsequently re-established a vegetative population. Implications for the bloom dynamics of this toxic dinoflagellate, a causative organism of paralytic shellfish poisoning, are discussed.     

SEXUAL PROCESSES IN THE LIFE CYCLE OF GYRODINIUM UNCATENUM (DINOPHYCEAE): A MORPHOGENETIC OVERVIEW1

Sexual processes in the life cycle of the dinoflagellate Gyrodinium uncatenum Hulburt were investigated in isolated field populations. Morphological and morphogenetic aspects of gamete production, planozygote formation, encystment, excystment, and planomeiocyte division are described from observations of living specimens, Protargol silver impregnated material and scanning electron microscope preparations. The sexual cycle was initiated by gamete formation which involved two asexual divisions of the vegetative organism. Gametes were fully differentiated following the second division and immediately capable of forming pairs. Either isogamous or anisogamous pairs were formed by the mid-ventral union of gametes. Gametes invariably joined with flagellar bases in close juxtaposition. Complete fusion of gametes required ca. 1 h, involved plasmogamy followed by karyogamy and resulted in a quadriflagellated planozygote. Planozygotes encysted in 24–48 h to yield a hypnozygote capable of overwintering in estuarine sediments. Hypnozygotes collected from sediment in late winter readily excysted upon exposure to temperatures above 15°C. A single quadriflagellated planomeiocyte emerged from the cyst and under culture conditions divided one to two days later. The four flagella were not evenly distributed at the first division and both bi- and tri-flagellated daughter cells were formed.     

Comparative study of the life cycles of Alexandrium tamutum and Alexandrium minutum (Gonyaulacales,Dinophyceae) in culture1

The microalgal genus Alexandrium includes species known to produce paralytic shellfish poisoning (PSP). Due to the importance of discriminating between HAB‐forming species, we compared the undescribed life‐cycle pattern of Alexandrium tamutum Montresor, Beran et U. John and of its toxic relative Alexandrium minutum Halim. Sexual stages, asexual and sexual division, mating type, and nuclear morphology were studied in both species. Sexual cysts are known to be morphologically identical. However, the relative size of the U‐shaped nucleus may be used to differentiate between the cysts of these species since DNA packaging in the resting cysts was lower in A. tamutum than in A. minutum, species in which the planozygote nucleus was reduced to half its volume prior to encystment. The dormancy period of the cysts was <20 d for A. tamutum, but longer than 1 month for A. minutum. In both species, cyst appearance needed to be explained by the existence of more than two sexual types (+/–), which indicates a complex heterothallic mating type. However, planozygotes of both species may divide instead of encysting. This characteristic was used for nutritional and heritage studies. Isolated planozygotes of both species encysted in larger percentages in medium deficient in both nitrates and phosphates (L/15) than in medium without phosphates added (L‐P), a medium in which most planozygotes neither divide nor encyst. Parental strains of A. minutum with and without the ventral pore formed planozygotes and, later, offspring with the ventral pore, although apparently smaller than usual. A synchronization–flow cytometry method for discriminating diploids formed by sexual fusion (planozygotes) from cells with 2C DNA content resulting from self‐duplication of DNA (dividing cells) was described. The results indicated that the maximum percentage of A. minutum planozygotes (20%) was achieved only 3 to 5 d after crossing the parental strains, and that light might not be needed for the sexual fusion and formation of planozygotes.     

Sexual and Asexual Processes in Protoperidinium steidingerae Balech (Dinophyceae), with Observations on Life-History Stages of Protoperidinium depressum (Bailey) Balech (Dinophyceae)

ABSTRACT. A suite of morphological, histological, and molecular techniques was used to reveal for the first time division, sexuality, mandatory dormancy period of hypnozygotes, and identity of life-history stages of any Protoperidinium spp. In both Protoperidinium steidingerae and Protoperidinium depressum , asexual division occurred by eleutheroschisis within a temporary cyst, yielding two daughter cells. Daughter cells were initially round and one-half to two-thirds the size of parent cells then rapidly increased in size, forming horns before separating. Gamete production and fusion was constitutive in clonal and non-clonal cultures, indicating that both species may be homothallic. Gametes were isogamous, approximately half the size and lacking the pink pigmentation of the vegetative cells, and were never observed to feed. Gamete fusion resulted in a planozygote with two longitudinal flagella. Planozygotes of P. steidingerae formed hypnozygotes. The fate of planozygotes of P. depressum is unknown. Hypnozygotes of P. steidingerae had a mandatory dormancy period of ca. 70 days. Germination resulted in planomeiocytes with two longitudinal flagella. Nuclear cyclosis occurred in the planozygotes of P. depressum , but in the planomeiocytes of P. steidingerae . The plate tabulation and gross morphology of gametes of P. steidingerae and P. depressum differed markedly from those of vegetative cells. Thus, misidentification of morphologically distinct life-history stages and incomplete examination of thecal plate morphology in field specimens has likely led to taxonomic confusion of Protoperidinium spp. in previous studies.     

EVIDENCE FOR ASEXUAL RESTING CYSTS IN THE LIFE CYCLE OF THE MARINE PERIDINOID DINOFLAGELLATE,SCRIPPSIELLA HANGOEI1

Scrippsiella hangoei (Schiller) Larsen is a peridinoid dinoflagellate that grows during winter and spring in the Baltic Sea. In culture this species formed round, smooth cysts when strains were mixed, indicating heterothallic sexuality and hypnozygote production. However, cysts of the same morphology were also formed in clonal strains exposed to slightly elevated temperature. To better understand the role of cysts in the life cycle of S. hangoei, cyst formation and dormancy were examined in culture experiments and the cellular DNA content of flagellate cells and cysts was compared in clonal and mixed strains using flow cytometry. S. hangoei exhibited a high rate of cyst formation in culture. Cysts produced in both clonal and mixed strain cultures were thick‐walled and underwent a dormancy period of 4 months before germinating. The S. hangoei flagellate cell population DNA distributions consisted of 1C, intermediate, and 2C DNA, indicative of respective eukaryotic cell cycle phases G1, S, and G2M. The majority (>95%) of cysts had a measured DNA content equivalent to the lower 1C DNA value, indicating a haploid nuclear phase and an asexual mode of cyst formation. A small percentage (<5%) of cysts produced in the mixed strain culture had 2C DNA, and thus could have been diploid zygotes. These findings represent the first measurements of dinoflagellate resting cyst DNA content, and provide the first quantitative evidence for dinoflagellate asexual resting cysts. Asexual resting cysts may be a more common feature of dinoflagellate life cycles than previously thought.     

EFFECTS OF PARENTAL FACTORS AND MEIOSIS ON SEXUAL OFFSPRING OF GYMNODINIUM NOLLERI (DINOPHYCEAE)1

Clonal strains of the dinoflagellate Gymnodinium nolleri Ellegaard and Moestrup were intercrossed to determine if cyst‐related traits are genetically regulated and to clarify unknown aspects in the sexuality of this species. The objectives were to determine whether the parental identity influenced the physiological and morphological aspects of the cyst offspring, and to describe and compare nuclear development and cell division of encysting and non‐encysting zygotes. Variables characteristic of each parental cross (difference in growth rates among parents, cyst production (CP), and genetic distance (GD) among parents assessed via an amplified fragment length analysis analysis) were studied to seek for possible relationships of the parental crosses with some characteristics of the cyst offspring (cyst size, length of dormancy period, germination success, and germling viability (V)). A principal component analysis using these variables showed three main results: (1) the dormancy period of cysts responded to a simple pattern of inheritance, (2) the larger the GD between parents, the smaller the CP, and progeny V, and (3) the size of cysts was influenced by both CP and the parental strain identity. A stable inheritance of the short dormancy period (14.6±5.5 days), dominant over medium (31.0±8.5 days) and long periods (52.7±9.2 days), was confirmed through two subsequent generations of cysts. The regulation of the sexual processes by a multiple loci system is discussed based on the pattern of inheritance of the dormancy period and the number of sexual recombination events recorded within cultures with self‐CP capability. Fusion of the gamete nuclei happened 0–48 h after the total cytoplasmic fusion. The nucleus of the zygote was bilobed and had thick and distinct chromosomes. Similar processes of nuclear and cell division occurred in the non‐encysting or encysting planozygote, and were characterized by the loss of the chromosomal structure, an apparent increase of the DNA content, and the formation of thinner chromosomes.     

INTERACTIVE EFFECTS OF SALINITY AND TEMPERATURE ON PLANOZYGOTE AND CYST FORMATION OF ALEXANDRIUM MINUTUM (DINOPHYCEAE) IN CULTURE1

The factors regulating dinoflagellate life‐cycle transitions are poorly understood. However, their identification is essential to unravel the causes promoting the outbreaks of harmful algal blooms (HABs) because these blooms are often associated with the formation and germination of sexual cysts. Nevertheless, there is a lack of knowledge on the factors regulating planozygote‐cyst transitions in dinoflagellates due to the difficulties of differentiating planozygotes from vegetative stages. In the present study, two different approaches were used to clarify the relevance of environmental factors on planozygote and cyst formation of the toxic dinoflagellate Alexandrium minutum Halim. First, the effects of changes in initial phosphate (P) and nitrate (N) concentrations in the medium on the percentage of planozygotes formed were examined using flow cytometry. Second, two factorial designs were used to determine how salinity (S), temperature (T), and the density of the initial cell inoculum (I) affect planozygote and resting‐cyst formation. These experiments led to the following conclusions: 1. Low P/N ratios seem to induce gamete expression because the percentage of planozygotes recorded in the absence of added phosphate (‐P) was significantly higher than that obtained in the absence of added nitrogen (‐N), or when the concentrations of both nitrogen and phosphate were 20 times lower (N/20 + P/20). 2. Salinity (S) and temperature (T) strongly affected both planozygote and cyst formation, as sexuality in the population increased significantly as salinity decreased and temperatures increased. S, T combinations that resulted in no significant cyst formation were, however, favorable for vegetative growth, ruling out the possibility of negative effects on cell physiology. 3. The initial cell density is thought to be important for sexual cyst formation by determining the chances of gamete contact. However, the inoculum concentrations tested did not explain either planozygote formation or the appearance of resting cysts.     

Evidence for Production of Sexual Resting Cysts by the Toxic Dinoflagellate Karenia mikimotoi in Clonal Cultures and Marine Sediments

The toxic dinoflagellate Karenia mikimotoi has been well-known for causing large-scale and dense harmful algal blooms (HABs) in coastal waters worldwide and serious economic loss in aquaculture and fisheries and other adverse effects on marine ecosystems. Whether K. mikimotoi forms resting cysts has been a puzzling issue regarding to the mechanisms of bloom initiation and geographic expansion of this species. We provide morphological and molecular confirmation of sexually produced thin-walled resting cysts by K. mikimotoi based on observations of laboratory cultures and their direct detection in marine sediments. Light and scanning electron microscopy evidences for sexual reproduction include attraction and pairing of gametes, gamete fusion, formation of planozygote and thin-walled cyst, and the documentation of the thin-walled cyst germination processes. Evidence for cysts in marine sediments was in three aspects: positive PCR detection of cysts using species-specific primers in the DNA extracted from whole sediments; fluorescence in situ hybridization detection of cysts using FISH probes; and single-cell PCR sequencing for cysts positively labeled with FISH probes. The existence of sexually produced, thin-walled resting cysts by K. mikimotoi provides a possible mechanism accounting for the initiation of annually recurring blooms at certain regions and global expansion of the species during the past decades.     

The toxic dinoflagellate Cochlodinium polykrikoides (Dinophyceae) produces resting cysts

While harmful algal blooms (HABs) caused by the toxic dinoflagellate Cochlodinium polykrikoides have been known to science for more than a century, the past two decades have witnessed an extraordinary expansion of these events across Asia, North America, and even Europe. Although the production of resting cysts and subsequent transport via ships’ ballast water or/and the transfer of shellfish stocks could facilitate this expansion, confirmative evidence for cyst production by C. polykrikoides is not available. Here, we provide visual confirmation of the production of resting cysts by C. polykrikoides in laboratory cultures isolated from North America. Evidence includes sexually mating cell pairs, planozygotes with two longitudinal flagella, formation of both pellicular (temporary) cysts and resting cysts, and a time series of the cyst germination process. Resting cyst germination occurred up to 1 month after cyst formation and 2–40% of resting cysts were successfully germinated in cultures maintained at 18–21 °C. Pellicular cysts with hyaline membranes were generally larger than resting cysts, displayed discernable cingulum and/or sulcus, and reverted to vegetative cells within 24 h to ∼1 week of formation. A putative armored stage of C. polykrikoides was not observed during any life cycle stage in this study. This definitive evidence of resting cyst production by C. polykrikoides provides a mechanism to account for the recurrence of annual blooms in given locales as well as the global expansion of C. polykrikoides blooms during the past two decades.     

Life history,excystment features,and growth characteristics of the Mediterranean harmful dinoflagellate Alexandrium pseudogonyaulax

Studies considering the biology and ecology of the toxic bloom‐forming species, Alexandrium pseudogonyaulax, are rare. Our results highlight five features not described before in A. pseudogonyaulax life cycle: (i) A. pseudogonyaulax gametes showed two modes of conjugation, anisogamy and isogamy, (ii) sexual conjugation occurs either in the dark or in the light phase by engulfment or a fusion process, (iii) the presence of planozygote and newly formed cysts in monoclonal culture suggests homothallism, (iv) newly formed cysts have very dark vesicular content and are mostly unparatabulated when observed under light microscope and (v) natural resting cysts are able to give either a planomeiocyte or two vegetative cells. Cyst viability was enhanced after 5 months of cold storage (4°C), with excystment rate reaching 97% after 3 d of incubation. Excystment rate was highest (43%–79%) in Enriched Natural Sea Water diluted culture medium, whereas few germling cells were able to survive without the culture medium (0%–13%). Salinity‐irradiance experiments revealed that the highest cell concentrations occur at high irradiances for all the tested salinities. Vegetative growth rates generally increased with increasing irradiance, and were less dependent on salinity variations. The relatively low growth rate, low cell densities in the laboratory, and the notable capacity of producing cysts along growth phases of A. pseudogonyaulax could explain the occurrence of high resting cysts densities in the sediment of Bizerte lagoon and the relatively low abundances of vegetative cells in the water column.     

ULTRASTRUCTURE AND LSU RDNA‐BASED PHYLOGENY OF ESOPTRODINIUM GEMMA (DINOPHYCEAE), WITH NOTES ON FEEDING BEHAVIOR AND THE DESCRIPTION OF THE FLAGELLAR BASE AREA OF A PLANOZYGOTE1

A small, freshwater dinoflagellate with an incomplete cingulum, identified as Esoptrodinium gemma Javornický (=Bernardinium bernardinense sensu auctt. non sensu Chodat), was maintained in mixed culture and examined using light and serial section TEM. Vegetative flagellate cells, large cells with two longitudinal flagella (planozygotes), and cysts were examined. The cells displayed a red eyespot near the base of the longitudinal flagellum, made of two or three layers of pigment globules not bounded by a membrane. Yellow‐green, band‐shaped chloroplasts, bounded by three membranes and containing lamella with three thylakoids, were present in both flagellate cells and cysts. Most cells had food vacuoles, containing phagotrophically ingested chlamydomonads or chlorelloid green algae; ingestion occurred through the ventral area, involving a thin pseudopod apparently driven by the peduncle. The pusule was tubular, with numerous diverticula in its distal portion, and opened into the longitudinal flagellar canal. Three roots were associated with each pair of flagellar bases, both in vegetative cells and in a planozygote. The longitudinal microtubular root bifurcated around the longitudinal basal body. The planozygote contained a single peduncle and associated structures, and a single transverse flagellar canal with the two converging transverse flagella. Using two ciliates as outgroup species, phylogenetic analyses based on maximum parsimony, neighbor‐joining and posterior probability (Bayesian analysis) supported a clade comprising Esoptrodinium, Tovellia, and Jadwigia.     

Life cycle stages of the benthic palytoxin-producing dinoflagellate Ostreopsis cf. ovata (Dinophyceae)

The asexual and sexual reproduction of Ostreopsis cf. ovata was studied in the field and in cultures isolated from two locations in the Mediterranean Sea. Asexual division took place in the motile stage by the sharing of theca (desmoschisis). High cell-size variability and differences in division capability were detected in the cultures. Thecal analyses and nuclear division patterns allowed characterization of the different phases of dividing cells obtained during an in situ cell-cycle sampling performed off Llavaneres beach (Northeast Spain). During the 45-h cycle, binucleated cells accounted for 2.6% of the population. Division was initiated with the onset of dusk and reached a maximum 3–4 h before dawn. No dividing cells were detected after 09:00 AM. Sexuality occurred both in cultures and in natural populations of O. cf. ovata. Mating gamete pairs were the only sexual stages that could be distinguished from vegetative stages. The differences between these pairs and dividing cells are described herein. None of the individually isolated gamete pairs underwent fusion nor encystment, instead, in most of them one member of the gamete pair divided. Rather than plasmogamy, there was evidence of nuclear migration from one gamete to the other. Pellicle cysts, thin-walled cysts, and thecate cysts were also identified and studied. These cysts, embedded in abundant mucilage in a bloom-derived incubated sample, were able to germinate for as long as 6 months after their formation. Our results suggest that they constitute the overwintering population that causes recurrent blooms of O. cf. ovata in some areas of the Mediterranean Sea.