Multigene phylogeny reveals a cryptic diversity in the genus Dinobryon (Chrysophyceae) with integrative description of five new species.

Introduction: The genus Dinobryon is one of the most recognizable chrysophyte genera, characterized by dendroid colonies with a biflagellate inside each cellulosic lorica. The representative forms of lorica are cylindrical, conical, vase, or funnel shaped, with undulation on the lorica wall. Traditionally, the morphological characteristics of the lorica and the colony organization have been used for the delimitation of Dinobryon species. Methods: To understand the taxonomy and phylogeny of colonial Dinobryon species, we performed molecular and morphological studies using 39 unialgal cultures and 46 single colony isolations from environmental specimens collected in Korea. We used a nuclear internal transcript spacer (ITS1-5.8S-ITS2) to find the genetic diversity of Dinobryon from environmental samples and a combined dataset from six gene sequences (nuclear SSU and LSU rRNA, plastid LSU rRNA, rbcL and psaA, and mitochondrial CO1 genes) for phylogenetic analysis. Results and discussion: We found 15 different lineages based on the genetic diversity of the nuclear ITS sequences. The phylogenetic tree of the colonial species based on the combined multigene dataset were divided into 18 subclades, including five new species, each with unique molecular signatures for the E23-5 helix of the V4 region in the nuclear SSU rRNA and the E11-1 helix of D7b, and the E20-1 helix of D8 regions in the nuclear LSU rRNA. Morphological studies were focused on lorica dimension and shape, and stomatocyst morphology. The Dinobryon species showed similarities or differences in lorica morphologies between and within species, and also differences in lorica size between culture and environmental samples. Five Dinobryon species formed distinctive stomatocysts, their stomatocyst morphologies, including collar structure, surface ornamentation, and cyst shape, showed unique characteristics in each species and were useful for identification. Here, we propose five new species based on morphological and molecular evidences: D. cylindricollarium, D. exstoundulatum, D. inclinatum, D. similis, and D. spinum.

Gene loss, pseudogenization, and independent genome reduction in non-photosynthetic species of Cryptomonas (Cryptophyceae) revealed by comparative nucleomorph genomics.

BACKGROUND: Cryptophytes are ecologically important algae of interest to evolutionary cell biologists because of the convoluted history of their plastids and nucleomorphs, which are derived from red algal secondary endosymbionts. To better understand the evolution of the cryptophyte nucleomorph, we sequenced nucleomorph genomes from two photosynthetic and two non-photosynthetic species in the genus Cryptomonas. We performed a comparative analysis of these four genomes and the previously published genome of the non-photosynthetic species Cryptomonas paramecium CCAP977/2a. RESULTS: All five nucleomorph genomes are similar in terms of their general architecture, gene content, and gene order and, in the non-photosynthetic strains, loss of photosynthesis-related genes. Interestingly, in terms of size and coding capacity, the nucleomorph genome of the non-photosynthetic species Cryptomonas sp. CCAC1634B is much more similar to that of the photosynthetic C. curvata species than to the non-photosynthetic species C. paramecium. CONCLUSIONS: Our results reveal fine-scale nucleomorph genome variation between distantly related congeneric taxa containing photosynthetic and non-photosynthetic species, including recent pseudogene formation, and provide a first glimpse into the possible impacts of the loss of photosynthesis on nucleomorph genome coding capacity and structure in independently evolved colorless strains.

Evolutionary Dynamics and Lateral Gene Transfer in Raphidophyceae Plastid Genomes.

The Raphidophyceae is an ecologically important eukaryotic lineage of primary producers and predators that inhabit marine and freshwater environments worldwide. These organisms are of great evolutionary interest because their plastids are the product of eukaryote-eukaryote endosymbiosis. To obtain deeper insight into the evolutionary history of raphidophycean plastids, we sequenced and analyzed the plastid genomes of three freshwater and three marine species. Our comparison of these genomes, together with the previously reported plastid genome of Heterosigma akashiwo, revealed unexpected variability in genome structure. Unlike the genomes of other analyzed species, the plastid genome of Gonyostomum semen was found to contain only a single rRNA operon, presumably due to the loss of genes from the inverted repeat (IR) region found in most plastid genomes. In contrast, the marine species Fibrocapsa japonica contains the largest IR region and overall plastid genome for any raphidophyte examined thus far, mainly due to the presence of four large gene-poor regions and foreign DNA. Two plastid genes, tyrC in F. japonica and He. akashiwo and serC in F. japonica, appear to have arisen via lateral gene transfer (LGT) from diatoms, and several raphidophyte open reading frames are demonstrably homologous to sequences in diatom plasmids and plastid genomes. A group II intron in the F. japonica psbB gene also appears to be derived by LGT. Our results provide important insights into the evolutionary history of raphidophyte plastid genomes via LGT from the plastids and plasmid DNAs of diatoms.

Molecular Phylogeny and Taxonomy of the Genus Spumella (Chrysophyceae) Based on Morphological and Molecular Evidence.

The genus Spumella, established by Cienkowsky in 1870, is characterized by omnivory, two (rarely three) flagella, a short stick-like structure beneath the flagella, a threadlike stalk, cell division via constriction and cyst formation. Since the first phylogenetic study of Spumella-like flagellates, their paraphyly has consistently been shown, with separation into several genera. More recently, Spumella was carefully investigated using molecular and morphological data to propose seven new species. Classification of this genus and knowledge of its species diversity remain limited because Spumella-like flagellates are extremely difficult to identify based on limited morphological characters. To understand the phylogeny and taxonomy of Spumella, we analyzed molecular and morphological data from 47 strains, including 18 strains isolated from Korean ponds or swamps. Nuclear SSU, ITS and LSU rDNA data were used for maximum likelihood and Bayesian analyses. The molecular data divided the strains into 15 clades, including seven new lineages, each with unique molecular signatures for nuclear SSU rRNA from the E23-2 to E23-5 domains, the spacer between the E23-8 and E23-9 domains of the V4 region and domain 29 of the V5 region. Our results revealed increased species diversity in Spumella. In contrast to the molecular phylogeny results, the taxa showed very similar cell morphologies, suggesting morphological convergence into simple nanoflagellates to enable heterotrophy. Three new species produced stomatocysts in culture. Aspects of stomatocyst morphology, including collar structure, surface ornamentation, and cyst shape, were very useful in differentiating the three species. The general ultrastructure of Spumella bureschii strain Baekdongje012018B8 and S. benthica strain Hwarim032418A5 showed the typical chrysophyte form for the leucoplast, a vestigial chloroplast surrounded by four envelope membranes, supporting the hypothesis that Spumella evolved from a phototroph to a heterotroph via the loss of its photosynthetic ability. Seven new species are proposed: S. benthica, S. communis, S. longicolla, S. oblata, S. rotundata, S. similis, and S. sinechrysos.

Comparative Plastid Genomics of Non-Photosynthetic Chrysophytes: Genome Reduction and Compaction.

Spumella-like heterotrophic chrysophytes are important eukaryotic microorganisms that feed on bacteria in aquatic and soil environments. They are characterized by their lack of pigmentation, naked cell surface, and extremely small size. Although Spumella-like chrysophytes have lost their photosynthetic ability, they still possess a leucoplast and retain a plastid genome. We have sequenced the plastid genomes of three non-photosynthetic chrysophytes, Spumella sp. Baeckdong012018B8, Pedospumella sp. Jangsampo120217C5 and Poteriospumella lacustris Yongseonkyo072317C3, and compared them to the previously sequenced plastid genome of "Spumella" sp. NIES-1846 and photosynthetic chrysophytes. We found the plastid genomes of Spumella-like flagellates to be generally conserved with respect to genome structure and housekeeping gene content. We nevertheless also observed lineage-specific gene rearrangements and duplication of partial gene fragments at the boundary of the inverted repeat and single copy regions. Most gene losses correspond to genes for proteins involved in photosynthesis and carbon fixation, except in the case of petF. The newly sequenced plastid genomes range from ~55.7 kbp to ~62.9 kbp in size and share a core set of 45 protein-coding genes, 3 rRNAs, and 32 to 34 tRNAs. Our results provide insight into the evolutionary history of organelle genomes via genome reduction and gene loss related to loss of photosynthesis in chrysophyte evolution.

Comparative plastid genomics of Synurophyceae: inverted repeat dynamics and gene content variation.

BACKGROUND: The Synurophyceae is one of most important photosynthetic stramenopile algal lineages in freshwater ecosystems. They are characterized by siliceous scales covering the cell or colony surface and possess plastids of red-algal secondary or tertiary endosymbiotic origin. Despite their ecological and evolutionary significance, the relationships amongst extant Synurophyceae are unclear, as is their relationship to most other stramenopiles. RESULTS: Here we report a comparative analysis of plastid genomes sequenced from five representative synurophycean algae. Most of these plastid genomes are highly conserved with respect to genome structure and coding capacity, with the exception of gene re-arrangements and partial duplications at the boundary of the inverted repeat and single-copy regions. Several lineage-specific gene loss/gain events and intron insertions were detected (e.g., cemA, dnaB, syfB, and trnL). CONCLUSIONS: Unexpectedly, the cemA gene of Synurophyceae shows a strong relationship with sequences from members of the green-algal lineage, suggesting the occurrence of a lateral gene transfer event. Using a molecular clock approach based on silica fossil record data, we infer the timing of genome re-arrangement and gene gain/loss events in the plastid genomes of Synurophyceae.

Feeding and grazing impact by the bloom-forming euglenophyte Eutreptiella eupharyngea on marine eubacteria and cyanobacteria.

The phototrophic euglenophyte Eutreptiella eupharyngea often causes blooms in the coastal waters of many countries, but its mode of nutrition has not been assessed. This species has previously been considered as exclusively auxotrophic. To explore whether E. eupharyngea is a mixotrophic species, the protoplasm of E. eupharyngea cells were examined using light, epifluorescence, and transmission electron microscopy after eubacteria, the cyanobacterium Synechococcus sp., and diverse algal species were provided as potential prey. Furthermore, the ingestion rates of E. eupharyngea KR on eubacteria or Synechococcus sp. as a function of prey concentration were measured. In addition, grazing by natural populations of euglenophytes on natural populations of eubacteria in Masan Bay was investigated. This study is the first to report that E. eupharyngea is a mixotrophic species. Among the potential prey organisms offered, E. eupharyngea fed only on eubacteria and Synechococcus sp., and the maximum ingestion rates of these two organisms measured in the laboratory were 5.7 and 0.7 cells predator-1 h-1, respectively. During the field experiments, the maximum ingestion rates and grazing impacts of euglenophytes, including E. eupharyngea, on natural populations of eubacteria were 11.8 cells predator-1 h-1 and 1.228 d-1, respectively. Therefore, euglenophytes could potentially have a considerable grazing impact on marine bacterial populations.

Comparative mitochondrial genomics of cryptophyte algae: gene shuffling and dynamic mobile genetic elements.

BACKGROUND: Cryptophytes are an ecologically important group of algae comprised of phototrophic, heterotrophic and osmotrophic species. This lineage is of great interest to evolutionary biologists because their plastids are of red algal secondary endosymbiotic origin. Cryptophytes have a clear phylogenetic affinity to heterotrophic eukaryotes and possess four genomes: host-derived nuclear and mitochondrial genomes, and plastid and nucleomorph genomes of endosymbiotic origin. RESULTS: To gain insight into cryptophyte mitochondrial genome evolution, we sequenced the mitochondrial DNAs of five species and performed a comparative analysis of seven genomes from the following cryptophyte genera: Chroomonas, Cryptomonas, Hemiselmis, Proteomonas, Rhodomonas, Storeatula and Teleaulax. The mitochondrial genomes were similar in terms of their general architecture, gene content and presence of a large repeat region. However, gene order was poorly conserved. Characteristic features of cryptophyte mtDNAs included large syntenic clusters resembling α-proteobacterial operons that encode bacteria-like rRNAs, tRNAs, and ribosomal protein genes. The cryptophyte mitochondrial genomes retain almost all genes found in many other eukaryotes including the nad, sdh, cox, cob, and atp genes, with the exception of sdh2 and atp3. In addition, gene cluster analysis showed that cryptophytes possess a gene order closely resembling the jakobid flagellates Jakoba and Reclinomonas. Interestingly, the cox1 gene of R. salina, T. amphioxeia, and Storeatula species was found to contain group II introns encoding a reverse transcriptase protein, as did the cob gene of Storeatula species CCMP1868. CONCLUSIONS: These newly sequenced genomes increase the breadth of data available from algae and will aid in the identification of general trends in mitochondrial genome evolution. While most of the genomes were highly conserved, extensive gene arrangements have shuffled gene order, perhaps due to genome rearrangements associated with hairpin-containing mobile genetic elements, tRNAs with palindromic sequences, and tandem repeat sequences. The cox1 and cob gene sequences suggest that introns have recently been acquired during cryptophyte evolution. Comparison of phylogenetic trees based on plastid and mitochondrial genome data sets underscore the different evolutionary histories of the host and endosymbiont components of present-day cryptophytes.

Deletion of the chloroplast LTD protein impedes LHCI import and PSI-LHCI assembly in Chlamydomonas reinhardtii.

Nuclear-encoded light-harvesting chlorophyll- and carotenoid-binding proteins (LHCPs) are imported into the chloroplast and transported across the stroma to thylakoid membrane assembly sites by the chloroplast signal recognition particle (CpSRP) pathway. The LHCP translocation defect (LTD) protein is essential for the delivery of imported LHCPs to the CpSRP pathway in Arabidopsis. However, the function of the LTD protein in Chlamydomonas reinhardtii has not been investigated. Here, we generated a C. reinhardtii ltd (Crltd) knockout mutant by using CRISPR-Cas9, a new target-specific knockout technology. The Crltd1 mutant showed a low chlorophyll content per cell with an unusual increase in appressed thylakoid membranes and enlarged cytosolic vacuoles. Profiling of thylakoid membrane proteins in the Crltd1 mutant showed a more severe reduction in the levels of photosystem I (PSI) core proteins and absence of functional LHCI compared with those of photosystem II, resulting in a much smaller PSI pool size and diminished chlorophyll antenna size. The lack of CrLTD did not prevent photoautotrophic growth of the cells. These results are substantially different from those for Arabidopsis ltd null mutant, indicating LTD function in LHCP delivery and PSI assembly may not be as stringent in C. reinhardtii as it is in higher plants.

Evolutionary Dynamics of Cryptophyte Plastid Genomes.

Cryptophytes are an ecologically important group of largely photosynthetic unicellular eukaryotes. This lineage is of great interest to evolutionary biologists because their plastids are of red algal secondary endosymbiotic origin and the host cell retains four different genomes (host nuclear, mitochondrial, plastid, and red algal nucleomorph). Here, we report a comparative analysis of plastid genomes from six representative cryptophyte genera. Four newly sequenced cryptophyte plastid genomes of Chroomonas mesostigmatica, Ch. placoidea, Cryptomonas curvata, and Storeatula sp. CCMP1868 share a number of features including synteny and gene content with the previously sequenced genomes of Cryptomonas paramecium, Rhodomonas salina, Teleaulax amphioxeia, and Guillardia theta. Our analysis of these plastid genomes reveals examples of gene loss and intron insertion. In particular, the chlB/chlL/chlN genes, which encode light-independent (dark active) protochlorophyllide oxidoreductase (LIPOR) proteins have undergone recent gene loss and pseudogenization in cryptophytes. Comparison of phylogenetic trees based on plastid and nuclear genome data sets show the introduction, via secondary endosymbiosis, of a red algal derived plastid in a lineage of chlorophyll-c containing algae. This event was followed by additional rounds of eukaryotic endosymbioses that spread the red lineage plastid to diverse groups such as haptophytes and stramenopiles.

Euglenophycin is produced in at least six species of euglenoid algae and six of seven strains of Euglena sanguinea.

Euglena sanguinea is known to produce the alkaloid toxin euglenophycin and is known to cause fish kills and inhibit mammalian tissue and microalgal culture growth. An analysis of over 30 species of euglenoids for accumulation of euglenophycin identified six additional species producing the toxin; and six of the seven E. sanguinea strains produced the toxin. A phylogenetic assessment of these species confirmed most taxa were in the Euglenaceae, whereas synthesis capability apparently has been lost in the Phacus, Eutreptiella, and Discoplastis branches.

Cryptophyte gene regulation in the kleptoplastidic, karyokleptic ciliate Mesodinium rubrum.

Photosynthesis in the ciliate Mesodinium rubrum is achieved using a consortium of cryptophyte algal organelles enclosed in its specialized vacuole. A time-series microarray analysis was conducted on the photosynthetic ciliate using an oligochip containing 15,654 primers designed from EST data of the cryptophyte prey, Teleaulax amphioxeia. The cryptophycean nuclei were transcriptionally active over 13 weeks and approximately 13.5% of transcripts in the ciliate came from the sequestered nuclei. The cryptophyte nuclei and chloroplasts could divide in the ciliate, which were loosely synchronized with host cell division. A large epigenetic modification occurred after the cryptophyte nuclei were sequestered into the ciliate. Most cryptophyte genes involved in the light and dark reactions of photosynthesis, chlorophyll assimilation, as well as in DNA methylation, were consistently up-regulated in the ciliate. The imbalance of division rate between the sequestered cryptophyte nuclei and host nuclei may be the reason for the eventual cessation of the kleptoplastidy.

The fate of cryptophyte cell organelles in the ciliate Mesodinium cf. rubrum subjected to starvation.

Mesodinium rubrum Lohmann is a mixotrophic ciliate and one of the best studied species exhibiting acquired phototrophy. To investigate the fate of cryptophyte organelles in the ciliate subjected to starvation, we conducted ultrastructural studies of a Korean strain of M. cf. rubrum during a 10 week starvation experiments. Ingested cells of the cryptophyte Teleaulax amphioxeia were first enveloped by ciliate membrane, and then prey organelles, including ejectisomes, flagella, basal bodies and flagellar roots, were digested. Over time, prey nuclei protruded into the cytoplasm of the ciliate, their size and volume increased, and their number decreased, suggesting that the cryptophyte nuclei likely fused with each other in the ciliate cytoplasm. At 4 weeks of starvation, M. cf. rubrum cells without cryptophyte nuclei started to appear. At 10 weeks of starvation, only two M. cf. rubrum cells still possessing a cryptophyte nucleus had relatively intact chloroplast-mitochondria complexes (CMCs), while M. cf. rubrum cells without cryptophyte nuclei had a few damaged CMCs. This is the first ultrastructural study demonstrating that cryptophyte nuclei undergo a dramatic change inside M. cf. rubrum in terms of size, shape, and number following their acquisition.

Do All Dinoflagellates have an Extranuclear Spindle?

The syndinean dinoflagellates are a diverse assemblage of alveolate endoparasites that branch basal to the core dinoflagellates. Because of their phylogenetic position, the syndineans are considered key model microorganisms in understanding early evolution in the dinoflagellates. Closed mitosis with an extranuclear spindle that traverses the nucleus in cytoplasmic grooves or tunnels is viewed as one of the morphological features shared by syndinean and core dinoflagellates. Here we describe nuclear morphology and mitosis in the syndinean dinoflagellate Amoebophrya sp. from Akashiwo sanguinea, a member of the A. ceratii complex, as revealed by protargol silver impregnation, DNA specific fluorochromes, and transmission electron microscopy. Our observations show that not all species classified as dinoflagellates have an extranuclear spindle. In Amoebophrya sp. from A. sanguinea, an extranuclear microtubule cylinder located in a depression in the nuclear surface during interphase moves into the nucleoplasm via sequential membrane fusion events and develops into an entirely intranuclear spindle. Results suggest that the intranuclear spindle of Amoebophrya spp. may have evolved from an ancestral extranuclear spindle and indicate the need for taxonomic revision of the Amoebophryidae.

The Plastid Genome of the Cryptomonad Teleaulax amphioxeia.

Teleaulax amphioxeia is a photosynthetic unicellular cryptophyte alga that is distributed throughout marine habitats worldwide. This alga is an important plastid donor to the dinoflagellate Dinophysis caudata through the ciliate Mesodinium rubrum in the marine food web. To better understand the genomic characteristics of T. amphioxeia, we have sequenced and analyzed its plastid genome. The plastid genome sequence of T. amphioxeia is similar to that of Rhodomonas salina, and they share significant synteny. This sequence exhibits less similarity to that of Guillardia theta, the representative plastid genome of photosynthetic cryptophytes. The gene content and order of the three photosynthetic cryptomonad plastid genomes studied is highly conserved. The plastid genome of T. amphioxeia is composed of 129,772 bp and includes 143 protein-coding genes, 2 rRNA operons and 30 tRNA sequences. The DNA polymerase III gene (dnaX) was most likely acquired via lateral gene transfer (LGT) from a firmicute bacterium, identical to what occurred in R. salina. On the other hand, the psbN gene was independently encoded by the plastid genome without a reverse transcriptase gene as an intron. To clarify the phylogenetic relationships of the algae with red-algal derived plastids, phylogenetic analyses of 32 taxa were performed, including three previously sequenced cryptophyte plastid genomes containing 93 protein-coding genes. The stramenopiles were found to have branched out from the Chromista taxa (cryptophytes, haptophytes, and stramenopiles), while the cryptophytes and haptophytes were consistently grouped into sister relationships with high resolution.

Assessing the evolutionary history of the class Synurophyceae (Heterokonta) using molecular, morphometric, and paleobiological approaches.

PREMISE OF THE STUDY: Heterokont algae of the class Synurophyceae, characterized by distinctive siliceous scales that cover the surface of the cell, are ecologically important in inland waters, yet their evolutionary history remains enigmatic. We explore phylogenetic relationships within this group of algae relative to geologic time, with a focus on evolution of siliceous components. METHODS: We combined an expansive five-gene and time-calibrated molecular phylogeny of synurophyte algae with an extensive array of fossil specimens from the middle Eocene to infer evolutionary trends within the group. KEY RESULTS: The group originated in the Jurassic approximately 157 million years ago (Ma), with the keystone genera Mallomonas and Synura diverging during the Early Cretaceous at 130 Ma. Mallomonas further splits into two major subclades, signaling the evolution of the V-rib believed to aid in the spacing and organization of scales on the cell covering. Synura also diverges into two primary subclades, separating taxa with forward-projecting spines on the scale from those with a keel positioned on the scale proper. Approximately one third of the fossil species are extinct, whereas the remaining taxa are linked to modern congeners. CONCLUSIONS: The taxonomy of synurophytes, which relies extensively on the morphology of the siliceous components, is largely congruent with molecular analyses. Scales of extinct synurophytes were significantly larger than those of modern taxa and may have played a role in their demise. In contrast, many fossil species linked to modern lineages were smaller in the middle Eocene, possibly reflecting growth in the greenhouse climatic state that characterized this geologic interval.

Ultrastructure and molecular phylogeny of Mesodinium coatsi sp. nov., a benthic marine ciliate.

Mesodinium is a globally distributed ciliate genus forming frequent and recurrent blooms in diverse marine habitats. Here, we describe a new marine species, Mesodinium coatsi n. sp., originally isolated from interstitial water of surface sand samples collected at Mohang Beach, Korea. The species was maintained under a mixotrophic growth condition for longer than 1 yr by providing a cryptomonad, Chroomonas sp., as the sole prey. Cell morphology and subcellular structure were examined by light microscopy, scanning, and transmission electron microscopy, and molecular phylogeny was inferred from nuclear-encoded 18S rDNA sequence data. Like other Mesodinium species, M. coatsi consisted of two hemispheres separated by two types of kinetids, and had tentacles located at the oral end of the cell. Several food vacuoles were observed in the cytoplasm, and partially digested prey cells sometimes existed in food vacuoles. Kinetids and the associated accessory structures were quite similar to those previously reported, but M. coatsi was differentiated from other marine Mesodinium species by ultrastructural characters of the dikinetids, polykinetids, and tentacles. We also provided a detailed illustration of infraciliature. Molecular phylogeny revealed that M. coatsi and Mesodinium chamaeleon were closely related to each other.

A rapid phenol toxicity test based on photosynthesis and movement of the freshwater flagellate, Euglena agilis Carter.

Phenol, a monosubstituted aromatic hydrocarbon with various commercial uses, is a major organic constituent in industrial wastewaters. The ecotoxic action of phenol for aquatic environment is well known. In this study, rapid phenol toxicity tests (1h) were developed based on chlorophyll a (Chl a) fluorescence and the movement parameters of the freshwater flagellate, Euglena agilis Carter. Phenol significantly reduced the maximum quantum yield (Fv/Fm) of photosystem II (PS II) and the maximum photosynthetic electron transport rate (rETRmax) with median effective concentration (EC50) values of 8.94 and 4.67 mM, respectively. Phenol reduced the motility and triggered change in the swimming velocity of the test organism. Among the parameters tested, velocity was the most sensitive biomarker with an EC50 of 3.17 mM. The EC50 values for Fv/Fm, motility, and velocity appear to overlap the permitted levels of phenol. In conclusion, the photosynthesis and movement of E. agilis can be fast and sensitive risk assessment parameters for the evaluation of phenol toxicity in municipal and industrial effluents.

The effect of starvation on plastid number and photosynthetic performance in the kleptoplastidic dinoflagellate Amylax triacantha.

The dinoflagellate Amylax triacantha is known to retain plastids of cryptophyte origin by engulfing the mixotrophic ciliate Mesodinium rubrum, itself a consumer of cryptophytes. However, there is no information on the fate of the prey's organelles and the photosynthetic performance of the newly retained plastids in A. triacantha. In this study, we conducted a starvation experiment to observe the intracellular organization of the prey's organelles and temporal changes in the photosynthetic efficiency of acquired plastids in A. triacantha. The ultrastructural observations revealed that while the chloroplast-mitochondria complexes and nucleus of cryptophyte were retained by A. triacantha, other ciliate organelles were digested in food vacuoles. Acquired plastids were retained in A. triacantha for about 1 mo and showed photosynthetic activities for about 18 d when measured by a pulse-amplitude modulation fluorometer.

Molecular Phylogeny and Cryptic Diversity of the Genus Phacus (Phacaceae, Euglenophyceae) and the Descriptions of Seven New Species.

The photosynthetic euglenoid genus Phacus is commonly found in freshwater; it is characterized by a rigid to semi-rigid cell, usually flat with numerous small discoid chloroplasts without pyrenoids. To understand the phylogenetic relationships among Phacus species, we used combined cytoplasmic SSU and LSU rDNA and plastid-encoded SSU and LSU rDNA sequence data from 82 strains, including seven Lepocinclis, three Discoplastis, one Eutreptia, and two Eutreptiella strains, as well as morphological data. The combined molecular dataset was analyzed using Bayesian and maximum likelihood methods. The resulting tree revealed that the genus Phacus was not monophyletic and fully resolved the phylogenetic relationships among eight lineages that were congruent with unique morphological characters in each clade. Molecular phylogeny and detailed morphological data led to the descriptions of seven new species: P. brevisulca, P. claviformis, P. hordei-formis, P. longisulca, P. minimus, P. paraorbicularis, and P. viridioryza. The new species were well supported as independent species and formed close relationships with small Phacus species and P. orbicularis in the tree. In addition, the new species had unique molecular signatures and showed high genetic diversity. Although the strains of P. orbicularis sensu Hübner were morphologically very similar, the phylogenetic analyses and genetic diversity suggested that P. orbicularis sensu Hübner should be divided into two subclades.