Genome of Halimeda opuntia reveals differentiation of subgenomes and molecular bases of multinucleation and calcification in algae.

Algae mostly occur either as unicellular (microalgae) or multicellular (macroalgae) species, both being uninucleate. There are important exceptions, however, as some unicellular algae are multinucleate and macroscopic, some of which inhabit tropical seas and contribute to biocalcification and coral reef robustness. The evolutionary mechanisms and ecological significance of multinucleation and associated traits (e.g., rapid wound healing) are poorly understood. Here, we report the genome of Halimeda opuntia, a giant multinucleate unicellular chlorophyte characterized by interutricular calcification. We achieve a high-quality genome assembly that shows segregation into four subgenomes, with evidence for polyploidization concomitant with historical sea level and climate changes. We further find myosin VIII missing in H. opuntia and three other unicellular multinucleate chlorophytes, suggesting a potential mechanism that may underpin multinucleation. Genome analysis provides clues about how the unicellular alga could survive fragmentation and regenerate, as well as potential signatures for extracellular calcification and the coupling of calcification with photosynthesis. In addition, proteomic alkalinity shifts were found to potentially confer plasticity of H. opuntia to ocean acidification (OA). Our study provides crucial genetic information necessary for understanding multinucleation, cell regeneration, plasticity to OA, and different modes of calcification in algae and other organisms, which has important implications in reef conservation and bioengineering.

Active meiosis during dinoflagellate blooms: A 'sex for proliferation' hypothesis.

In dinoflagellates, sexual reproduction is best known to be induced by adverse environmental conditions and culminate in encystment for survival ('sex for encystment'). Although increasing laboratory observations indicate that sex can lead to production of vegetative cells bypassing encystment, the occurrence of this alternative pathway in natural populations and its ecological roles remain poorly understood. Here we report evidence that sex in dinoflagellates can potentially be an instrument for bloom proliferation or extension. By bloom metatranscriptome profiling, we documented elevated expression of meiosis genes in two evolutionarily distinct species (Prorocentrum shikokuense and Karenia mikimotoi) during bloom, a timing unexpected of the 'sex for encystment' scenario. To link these genes to meiosis, we induced encystment and cyst germination in the cyst-forming species Scrippsiella acuminata, and found that five of these genes were upregulated during cyst germination, when meiosis occurs. Integrating data from all three species revealed that SPO11, MND1, and DMC1 were likely common between cyst-forming and non-encysting sex in dinoflagellates. Furthermore, flow cytometric analyses revealed consecutive rounds of DNA halving during blooms of P. shikokuense and K. mikimotoi, evidencing meiosis. These data provided novel evidence that sexual reproduction in dinoflagellates might serve to promote cell proliferation, and along with the consequent enhancement of genetic diversity facilitating resistance against pathogens and environmental stress, to boost or extend a bloom ('sex for proliferation'). The putative meiosis-specific genes and insights reported here will prove to be helpful for rigorously testing the hypothesis and addressing whether the two modes of sex are genetically predisposed (i.e. species-specific) or environmentally induced (switchable within species), and if the latter what triggers the switch.

Energy metabolism and genetic information processing mark major transitions in the life history of Scrippsiella acuminata (Dinophyceae).

Many dinoflagellates perform sexual reproduction and form cysts as a life history strategy to survive adverse environmental conditions and seed annual harmful algal blooms (HABs). The molecular mechanisms underpinning the life stage transitions can provide clues about how key environmental factors induce encystment and initiation of a HAB but are still poorly understood. Here, we conducted an integrated physiological and transcriptomic study to unravel the mechanisms in Scrippsiella acuminata. We established a culture from a bloom, induced cyst formation, and divided the process into four life stages. Transcriptomic analysis of these stages revealed 19,900 differentially expressed genes (DEGs). The expression of genes related to photosynthesis was significantly up-regulated from vegetative stage to immature cyst stage, consistent with the marked increase in cell contents of energy-storing macromolecules (carbohydrates and lipids). When proceeding to resting cysts, most photosynthesis genes were down-regulated while "genetic information processing" related genes were up-regulated. Comparing germinating cysts with resting cysts revealed 100 DEGs involved in energy metabolism, indicating a high energy requirement of germination. In addition, the transition from germinating cysts to vegetative cells featured up-regulation of photosynthesis. Our results demonstrate that energy storage and consumption play a pivotal role in cyst formation and germination respectively and genetic information processing is crucial in cyst dormancy.

Phytate as a Phosphorus Nutrient with Impacts on Iron Stress-Related Gene Expression for Phytoplankton: Insights from the Diatom Phaeodactylum tricornutum.

Phytoplankton have evolved a capability to acquire phosphorus (P) from dissolved organic phosphorus (DOP) since the preferred form, dissolved inorganic phosphate (DIP, or Pi), is often limited in parts of the ocean. Phytic acid (PA) is abundantly synthesized in plants and rich in excreta of animals, potentially enriching the DOP pool in coastal oceans. However, whether and how PA can be used by phytoplankton are poorly understood. Here, we investigated PA utilization and underlying metabolic pathways in the diatom model Phaeodactylum tricornutum. The physiological results showed that P. tricornutum could utilize PA as a sole source of P nutrient to support growth. Meanwhile, the replacement of PA for DIP also caused changes in multiple cellular processes, such as inositol phosphate metabolism, photosynthesis, and signal transduction. These results suggest that PA is bioavailable to P. tricornutum and can directly participate in the metabolic pathways of PA-grown cells. However, our data showed that the utilization of PA was markedly less efficient than that of DIP, and PA-grown cells exhibited P and iron (Fe) nutrient stress signals. Implicated in these findings is the potential of complicated responses of phytoplankton to an ambient DOP species, which calls for more systematic investigation. IMPORTANCE PA is abundant in plants and cannot be digested by nonruminant animals. Hence, it is potentially a significant component of the DOP pool in coastal waters. Despite this potential importance, there is little information about its bioavailability to phytoplankton as a source of P nutrient and the molecular mechanisms involved. In this study, we found that part of PA could be utilized by the diatom P. tricornutum to support growth, and another portion of PA can act as a substrate directly participating in various metabolism pathways and cellular processes. However, our physiological and transcriptomic data show that PA-grown cells still exhibited signs of P stress and potential Fe stress. These results have significant implications in phytoplankton P nutrient ecology and provide a novel insight into multifaceted impacts of DOP utilization on phytoplankton nutrition and metabolism.

Differential enrichment and physiological impacts of ingested microplastics in scleractinian corals in situ.

Microplastics are emerging contaminants and widespread in the ocean, but their impacts on coral reef ecosystems are poorly understood, and in situ study is still lacking. In the present study, the distribution patterns of microplastics in the environment and inhabiting organisms were investigated along the east coast of Hainan Island, South China Sea, and the physiological impacts of the microplastics on scleractinian corals were analyzed. We documented average microplastic concentrations of 14.90 particlesL-1 in seawater, 343.04 particleskg-1 in sediment, 4.97 particlescm-2 in corals, and 0.67-3.12 particlescm-1 in Tridacnidae, Trochidae and fish intestines. Further analysis revealed that the characteristics of microplastics in the organisms were different from those in the environment, indicating preferential enrichment in the organisms. Furthermore, there was an obvious correlation between microplastic concentration and symbiotic density in corals. Furthermore, caspase3 activity was significantly positively correlated with the microplastic content in the small-polyp coral Pocillopora damicornis, but the large-polyp coral Galaxea fascicularis showed higher tolerance to microplastics. Taken together, our results suggest that microplastics are selectively enriched in corals and other reef-dwellers, in which they exact differential stress (apoptotic) effects, with the potential to impact the coral-Symbiodiniaceae symbiosis and alter the coral community structure.

Identification and expression analysis of meiosis-related genes in the harmful alga Heterosigma akashiwo (Raphidophyceae).

Sexual reproduction (SR), hallmarked by meiosis, is widespread in eukaryotes. In phytoplankton, SR has been observed in many lineages, but molecular information on SR or meiosis of harmful algal bloom (HAB) species is scarce. The raphidophyte Heterosigma akashiwo is a globally distributed and devastating HAB species, but molecular evidence of its SR or meiosis is lacking. Here, to address the gap of knowledge, the presence of meiotic genes in H. akashiwo were examined. Interestingly, seven meiosis-specific or related genes (SPO11, MND, RAD21, RAD51, MSH2, MSH6 and MEI2) were identified from H. akashiwo transcriptomes. Furthermore, expression patterns of these genes except MSH6 (excluded due to primer failure) were investigated using quantitative reverse-transcription PCR. Results showed that the examined genes exhibited similar diel rhythms, typically, highest in early dark period and then gradually decreasing until mid-day. In addition, the expression of these six genes was not higher in the stationary growth stage than in the exponential stage, as would be expected if meiosis was to form cysts, and their elevated expression in response to colchicine treatment (arresting cells in the G2/M transition) indicated a potential role of these genes in cell division and population growth in H. akashiwo. Consistent with this, we also found a morning to afternoon shift in the expression of these genes during the bloom of H. akashiwo. This study documents a part of the typical SR gene repertoire and its potential role in regulating cell division in H. akashiwo, offering candidates for population growth markers for bloom monitoring although its linkage to meiosis and SR remain to be studied further in the future.

Transcriptomic and physiological responses of Skeletonema costatum to ATP utilization.

The capacity of phytoplankton to utilize dissolved organic phosphorus (DOP) plays an important role in their competition for resources when the availability of dissolved inorganic phosphorus (DIP) is low in the aquatic systems. Here, we explored the physiological and molecular responses of a globally distributed marine diatom, Skeletonema costatum, in utilizing adenosine-5'-triphosphate (ATP) based on incubation experiments under ATP, DIP-replete, and DIP-depleted conditions. The results show that ATP supports the growth of S. costatum as efficiently as DIP. The pathway of S. costatum involved in utilizing ATP is not related to alkaline phosphatase (AP), an important DOP hydrolase, although extracellular hydrolysis is involved. The transcriptome analysis revealed several transcripts related to the hydrolase activity (e.g. NAD+ diphosphatase), which were significantly upregulated in the ATP culture group, indicating their possible involvement in ATP hydrolysis. Meanwhile, ATP-grown S. costatum exhibited downregulation of the genes related to a series of metabolic activities (e.g. purine metabolism), apparently to adapt to ATP condition.

RNA-seq profiling of Fugacium kawagutii reveals strong responses in metabolic processes and symbiosis potential to deficiencies of iron and other trace metals.

A healthy symbiotic relationship between corals and Symbiodiniaceae relies on suitable temperature and adequate nutrients including trace metals. Besides global warming, trace metal deficiency has been shown to cause coral bleaching, a phenomenon responsible for extensive coral reef degradation around the world. How trace metal deficiency impacts Symbiodiniaceae and coral symbiosis is poorly understood, however. In this study, we applied RNA-seq to investigate how Fugacium kawagutii responds to the deficiency of five trace metals (Fe2+, Zn2+, Cu2+, Mn2+, Ni2+). We identified 685 to 2805 differentially expressed genes (DEGs) from these trace metal deficiency conditions, among which 372 were commonly regulated by all the five trace metals and were significantly enriched in energy metabolism (e.g. fatty acid synthesis). Furthermore, genes associated with extracellular matrix (ECM), cell surface structure and cell adhesion were impacted, suggesting that the ability of recognition and adhesion of F. kawagutii may be altered by trace metal deficiencies. In addition, among the five metals, Fe2+ deficiency exhibited the strongest influence, with Fe-rich redox elements and many antioxidant synthesis genes being markedly down-regulated, indicative of adaptive reduction of Fe demand but a compromised ability to combat oxidative stress. Overall, deficiency of trace metals (especially Fe) seems to repress growth and ability of ROS scavenging, elevate energy metabolism and innate immunity, and alter cell adhesion capability, with implications in symbiosis disruption and coral bleaching.

Microplastic exposure represses the growth of endosymbiotic dinoflagellate Cladocopium goreaui in culture through affecting its apoptosis and metabolism.

Microplastics are widespread emerging marine pollutants that have been found in the coral reef ecosystem. In the present study, using Cladocopium goreaui as a symbiont representative, we investigated cytological, physiological, and molecular responses of a Symbiodiniaceae species to weeklong microplastic exposure (Polystyrene, diameter 1.0 µm, 9.0 × 109 particles L-1). The density and size of algal cells decreased significantly at 7 d and 6-7 d of microplastic exposure, respectively. Chlorophyll a content increased significantly at 7 d of exposure, whereas Fv/Fm did not change significantly during the entire exposure period. We observed significant increases in superoxide dismutase activity and caspase3 activation level, significant decrease in glutathione S-transferase activity, but no change in catalase activity during the whole exposure period. Transcriptomic analysis revealed 191 significantly upregulated and 71 significantly downregulated genes at 7 d after microplastic exposure. Fifteen GO terms were overrepresented for these significantly upregulated genes, which were grouped into four categories including transmembrane ion transport, substrate-specific transmembrane transporter activity, calcium ion binding, and calcium-dependent cysteine-type endopeptidase activity. Thirteen of the significantly upregulated genes encode metal ion transporter and ammonium transporter, and five light-harvesting protein genes were among the significantly downregulated genes. These results demonstrate that microplastics can act as an exogenous stressor, suppress detoxification activity, nutrient uptake, and photosynthesis, elevate oxidative stress, and raise the apoptosis level through upregulating ion transport and apoptotic enzymes to repress the growth of C. goreaui. These effects have implications in negative impacts of microplastics on coral-Symbiodiniaceae symbiosis that involves C. goreaui.

Circadian and irradiance effects on expression of antenna protein genes and pigment contents in dinoflagellate Prorocentrum donghaiense (Dinophycae).

PCP and acpPC are the two major antennae proteins that bind pigments in peridinin-containing dinoflagellates. The relationship between antennae proteins and cellular pigments at molecular level is still poorly understood. Here we identified and characterized the two antennae protein genes in dinoflagellate Prorocentrum donghaiense under different light conditions. The mature PCP protein was 32 kDa, while acpPC was a polyprotein each of 19 kDa. Both genes showed higher expression under low light than under high light, suggesting their possible role in a low light adaptation mechanism. The two genes showed differential diel expression rhythm, with PCP being more highly expressed in the dark than in the light period and acpPC the other way around. HPLC analysis of cellular pigments indicated a diel change of chlorophyll c2, but invariability of other pigments. A stable peridinin: chlorophyll a pigment ratio was detected under different light intensities and over the diel cycle, although the diadinoxanthin:chlorophyll a ratio increased significantly with light intensity. The results suggest that 1) PCP and acpPC genes are functionally distinct, 2) PCP and acpPC can function under low light as an adaptive mechanism in P. donghaiense, 3). the ratios of diadinoxanthin:chlorophyll a and peridinin: chlorophyll a can potentially be used as an indicator of algal photophysiological status and a pigment signature respectively under different light conditions in P. donghaiense.

Transcriptomic and physiological analyses of the dinoflagellate Karenia mikimotoi reveal non-alkaline phosphatase-based molecular machinery of ATP utilisation.

The ability to utilize dissolved organic phosphorus (DOP) is important for phytoplankton to survive the scarcity of dissolved inorganic phosphorus (DIP), and alkaline phosphatase (AP) has been the major research focus as a facilitating mechanism. Here, we employed a unique molecular ecological approach and conducted a broader search for underpinning molecular mechanisms of adenosine triphosphate (ATP) utilisation. Cultures of the dinoflagellate Karenia mikimotoi were set up in L1 medium (+P), DIP-depleted L1 medium (-P) and ATP-replacing-DIP medium (ATP). Differential gene expression was profiled for ATP and +P cultures using suppression subtractive hybridisation (SSH) followed by 454 pyrosequencing, and RT-qPCR methods. We found that ATP supported a similar growth rate and cell yield as L1 medium and observed DIP release from ATP into the medium, suggesting that K. mikimotoi cells were expressing extracellular hydrolases to hydrolyse ATP. However, our SSH, qPCR and enzymatic activity assays indicated that 5'-nucleotidase (5NT), rather than AP, was responsible for ATP hydrolysis. Further gene expression analyses uncovered that intercellular purine metabolism was significantly changed following the utilisation of ATP. Our findings reveal a multi-faceted machinery regulating ATP utilisation and P metabolism in K. mikimotoi, and underscore AP activity is not the exclusive indicator of DOP utilisation.

Morphological and molecular characterization of the toxic dinoflagellate Ostreopsis cf. ovata (Gonyaulacales: Dinophyceae) from Brazil (South Atlantic Ocean) / Caracterización morfológica y molecular del dinoflagelado tóxico Ostreopsis cf. ovata (Gonyaulacales: Dinophyceae) en Brasil (Océano Atlántico Sur)

AbstractOstreopsis cf. ovata is a toxic epiphytic dinoflagellate widely distributed in warm waters that often co-occur with species of the genera Coolia, Fukuyoa, Gambierdiscus and Prorocentrum. We investigated a strain isolated from the coast of Ubatuba, Brazil (Southwest Atlantic Ocean) by light and epifluorescence microscopies; we also report molecular data based on the LSU rDNA and ITS markers. Cells were 35-65 µm in the dorso-ventral diameter and 20-40 µm wide. We obtained the sequence of a ~1 900 base pair region of the rRNA gene cistron. In the LSU rDNA phylogeny, the sequences under the names O. ovata and O. cf. ovata branched into three clades. The ITS marker showed greater resolving power and the sequences of O. ovata/O. cf. ovata split into five clades. Our ITS sequence branched in a clade with sequences of strains from the Mediterranean Sea, European Atlantic coasts, subtropical NE Atlantic, other sequences from Brazil at Rio de Janeiro, and a few sequences from Japan. The cell dimensions and thecal plate arrangement were under the variability range reported in other ocean regions. Our observations confirm O. cf. ovata as the most commonly recorded species of Ostreopsis in the SW Atlantic Ocean. Ostreopsis cf. ovata co-occurred with Coolia malayensis in Brazil and Asia, but it has been commonly reported from the Mediterranean Sea, where C. malayensis has not yet been recorded; while Coolia malayensis has been reported from the Caribbean Sea, but not O. ovata. With the current knowledge, it is difficult to understand the factors that determine the biogeography of the tropical epiphytic dinoflagellates. Rev. Biol. Trop. 65 (3): 1022-1032. Epub 2017 September 01.

ResumenOstreopsis cf. ovata es un dinoflagelado tóxico epifítico de amplia distribución en aguas cálidas, que a menudo coincide con especies de los géneros Coolia, Fukuyoa, Gambierdiscus y Prorocentrum. Investigamos una cepa aislada en la costa de Ubatuba, Brasil (Atlántico sudoccidental) mediante microscopía óptica y de epifluorescencia. Obtuvimos una secuencia de una región de unos 1 900 pares de bases del cistrón del gen del ARN ribosómico. Las células tenían 35-65 µm de diámetro dorso-ventral y 20-40 µm de ancho. En la filogenia del marcador LSU rADN, las secuencias con los nombres O. ovata and O. cf. ovata se sitúan en tres grupos. El marcador ITS mostraba un mayor poder resolutivo y las secuencias de O. ovata/O. cf. ovata se separan en cinco grupos. Nuestra secuencia ITS se sitúa en un grupo con secuencias de cepas procedentes del Mar Mediterráneo, costas europeas Atlánticas, Atlántico subtropical nororiental, otras secuencias procedentes de Río de Janeiro en Brasil, y algunas secuencias de Japón. Las dimensiones celulares y la disposición de las placas tecales se sitúan en el rango de variabilidad descrito en otras regiones oceánicas. Nuestras observaciones confirman a O. cf. ovata como la especie más comúnmente registrada de Ostreopsis en el Atlántico sudoccidental. Ostreopsis cf. ovata coindice con Coolia malayensis en Brasil y Asia. Ostreopsis cf. ovata ha sido comúnmente encontrada en el Mar Mediterráneo, donde C. malayensis aún no ha sido registrada. Coolia malayensis has sido registrada en el Mar Caribe, donde O. ovata aún no ha sido encontrada. Es difícil comprender los factores que determinan la biogeografía de los dinoflagelados epífitos tropicales, a partir del conocimiento actual.

Morphological and molecular characterization of Ptychodiscus noctiluca revealed the polyphyletic nature of the order Ptychodiscales (Dinophyceae).

The planktonic dinoflagellate Ptychodiscus noctiluca combined distinctive morphological features such as a disk-shaped anteroposteriorly compressed cell body and an apical carina, together with a flexible and tough cell covering, suggesting intermediate characteristics between thecate and naked dinoflagellates. Ptychodiscus noctiluca was examined by light, epifluorescence, and scanning electron microscopy from specimens collected in the Mediterranean Sea and the North and South Atlantic Ocean. Ptychodiscus noctiluca showed a straight apical groove that bisected the carina, a cell covering with a polygonal surface reticulum, nucleus without capsule, sulcal intrusion in the episome, sulcal ventral flange, and yellowish-green chloroplasts that are shared characters with Brachidinium/Karenia. The cell division was the typical binary fission of gymnodinioid dinoflagellates, although exceptionally in an oblique transversal axis. We examined the intraspecific variability during incubation experiments. In the fattened cells, termed as Ptychodiscus carinatus, chloroplasts transformed into dark granules, and the cell acquired the swollen and smaller stage, termed as P. inflatus. Ptychodiscus carinatus, P. inflatus, and Diplocystis antarctica are synonyms of P. noctiluca. Molecular phylogeny based on the SSU rDNA sequence revealed that Ptychodiscus branched within the short-branching dinokaryotic dinoflagellates as an independent lineage with affinity to Brachidinium/Karenia and Karlodinium/Takayama in a generally poorly resolved clade. Our results indicated that the order Ptychodiscales, established for unarmored dinoflagellates with a strongly developed pellicle, has artificially grouped thecate dinoflagellates (Kolkwitziella, Herdmania), naked dinoflagellates with thick cell covering (Balechina, Cucumeridinium) and other insufficiently known unarmored genera with typical cell coverings (Brachidinium, Ceratoperidinium).

The Symbiodinium kawagutii genome illuminates dinoflagellate gene expression and coral symbiosis.

Dinoflagellates are important components of marine ecosystems and essential coral symbionts, yet little is known about their genomes. We report here on the analysis of a high-quality assembly from the 1180-megabase genome of Symbiodinium kawagutii. We annotated protein-coding genes and identified Symbiodinium-specific gene families. No whole-genome duplication was observed, but instead we found active (retro)transposition and gene family expansion, especially in processes important for successful symbiosis with corals. We also documented genes potentially governing sexual reproduction and cyst formation, novel promoter elements, and a microRNA system potentially regulating gene expression in both symbiont and coral. We found biochemical complementarity between genomes of S. kawagutii and the anthozoan Acropora, indicative of host-symbiont coevolution, providing a resource for studying the molecular basis and evolution of coral symbiosis.

Rhodopsin gene expression regulated by the light dark cycle, light spectrum and light intensity in the dinoflagellate Prorocentrum.

The proton pump rhodopsin is widely found in marine bacteria and archaea, where it functions to capture light energy and convert it to ATP. While found in several lineages of dinoflagellates, this gene has not been studied in Prorocentrales species and whether it functionally tunes to light spectra and intensities as in bacteria remains unclear. Here we identified and characterized this gene in the bloom-forming Prorocentrum donghaiense. It is a 7-helix transmembrane polypeptide containing conserved domains and critical amino acid residues of PPR. This gene is phylogenetically affiliated to the xanthorhodopsin clade, but seems to have a distinct evolutionary origin. Quantitative reverse transcription PCR showed that in regular cultures, the transcript abundance of the gene exhibited a clear diel pattern, high abundance in the light period and low in the dark. The same diel pattern was observed for protein abundance with a Western blot using specific antiserum. The rhythm was dampened when the cultures were shifted to continuous dark or light condition, suggesting that this gene is not under circadian clock control. Rhodopsin transcript and protein abundances varied with light intensity, both being highest at a moderate illumination level. Furthermore, the expression of this gene responded to different light spectra, with slightly higher transcript abundance under green than blue light, and lowest abundance under red light. Transformed Escherichia coli over-expressing this rhodopsin gene also exhibited an absorption maximum in the blue-green region with slightly higher absorption in the green. These rhodopsin-promoting light conditions are similar to the relatively turbid marine habitat where the species forms blooms, suggesting that this gene may function to compensate for the light-limited photosynthesis in the dim environment.

Fukuyoa paulensis gen. et sp. nov., a new genus for the globular species of the dinoflagellate Gambierdiscus (Dinophyceae).

The marine epiphytic dinoflagellate Gambierdiscus is a toxicologically important genus responsible for ciguatera fish poisoning, the principal cause of non-bacterial illness associated with fish consumption. The genus currently contains species exhibiting either globular or anterior-posteriorly compressed morphologies with marked differences in cell shape and plate arrangement. Here we report a third globular, epiphytic and tychoplanktonic species from the coasts of Ubatuba, Brazil. The new species can be distinguished from G. yasumotoi and G. ruetzleri by its broader first apical plate that occupies a larger portion of the epitheca. Accordingly, phylogenetic trees from small subunit (SSU) and large subunit (LSU) ribosomal DNA sequences also showed strongly supported separation of the new species from the G. yasumotoi/G. ruetzleri group albeit with short distance. The molecular phylogenies, which included new sequences of the planktonic species Goniodoma polyedricum, further indicated that the globular species of Gambierdiscus formed a tight clade, clearly separated (with strong bootstrap support) from the clade of lenticular species including the type for Gambierdiscus. The morphological and molecular data in concert support the split of Gambierdiscus sensu lato into two genera. Gambierdiscus sensu stricto should be reserved for the species with lenticular shapes, highly compressed anterioposteriorly, with short-shank fishhook apical pore plate, large 2' plate, low and ascending cingular displacement, and pouch-like sulcal morphology. The new genus name Fukuyoa gen. nov. should be applied to the globular species, slightly laterally compressed, with long-shank fishhook apical pore plate, large 1' plate, greater and descending cingular displacement, and not pouch-like vertically-oriented sulcal morphology. Fukuyoa contains the new species Fukuyoa paulensis gen. et sp. nov., and F. yasumotoi comb. nov. and F. ruetzleri comb. nov.

Effect of inorganic and organic ligands on the bioavailability of methylmercury as determined by using a mer-lux bioreporter.

A mer-lux bioreporter was constructed to assess the bioavailability of methylmercury [CH(3)Hg(II)] in Escherichia coli. The bioreporter was shown to be sensitive, with a detection limit of 2.5 nM CH(3)Hg(II), and was used to investigate the effects of chlorides, humic acids, and thiols on the bioavailability of CH(3)Hg(II) in E. coli. It was found that increasing the concentration of chlorides resulted in an increase in CH(3)Hg(II) bioavailability, suggesting that there was passive diffusion of the neutral complex (CH(3)HgCl(0)). Humic acids were found to reduce the bioavailability of CH(3)Hg(II) in varying degrees. Complexation with cysteine resulted in increased bioavailability of CH(3)Hg(II), while assays with equivalent concentrations of methionine and leucine had little or no effect on bioavailability. The mechanism of uptake of the mercurial-cysteine complexes is likely not passive diffusion but could result from the activities of a cysteine transport system. The bioavailability of CH(3)Hg(II) decreased with increasing glutathione concentrations.