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1.  Description of Two Species of Early Branching Dinoflagellates, Psammosa pacifica n. g., n. sp. and P. atlantica n. sp 
PLoS ONE  2012;7(6):e34900.
In alveolate evolution, dinoflagellates have developed many unique features, including the cell that has epicone and hypocone, the undulating transverse flagellum. However, it remains unclear how these features evolved. The early branching dinoflagellates so far investigated such as Hematodinium, Amoebophrya and Oxyrrhis marina differ in many ways from of core dinoflagellates, or dinokaryotes. Except those handful of well studied taxa, the vast majority of early branching dinoflagellates are known only by environmental sequences, and remain enigmatic. In this study we describe two new species of the early branching dinoflagellates, Psammosa pacifica n. g., n. sp. and P. atlantica n. sp. from marine intertidal sandy beach. Molecular phylogeny of the small subunit (SSU) ribosomal RNA and Hsp90 gene places Psammosa spp. as an early branch among the dinoflagellates. Morphologically (1) they lack the typical dinoflagellate epicone–hypocone structure, and (2) undulation in either flagella. Instead they display a mosaïc of dinokaryotes traits, i.e. (3) presence of bi-partite trychocysts; Oxyrrhis marina–like traits, i.e. (4) presence of flagellar hairs, (5) presence of two-dimensional cobweb scales ornamenting both flagella (6) transversal cell division; a trait shared with some syndineansand Parvilucifera spp. i.e. (7) a nucleus with a conspicuous nucleolus and condensed chromatin distributed beneath the nuclear envelope; as well as Perkinsus marinus -like features i.e. (8) separate ventral grooves where flagella emerge and (9) lacking dinoflagellate-type undulating flagellum. Notably Psammosa retains an apical complex structure, which is shared between perkinsids, colpodellids, chromerids and apicomplexans, but is not found in dinokaryotic dinoflagellates.
PMCID: PMC3377698  PMID: 22719825
2.  Extensive frameshift at all AGG and CCC codons in the mitochondrial cytochrome c oxidase subunit 1 gene of Perkinsus marinus (Alveolata; Dinoflagellata) 
Nucleic Acids Research  2010;38(18):6186-6194.
Diverse mitochondrial (mt) genetic systems have evolved independently of the more uniform nuclear system and often employ modified genetic codes. The organization and genetic system of dinoflagellate mt genomes are particularly unusual and remain an evolutionary enigma. We determined the sequence of full-length cytochrome c oxidase subunit 1 (cox1) mRNA of the earliest diverging dinoflagellate Perkinsus and show that this gene resides in the mt genome. Apparently, this mRNA is not translated in a single reading frame with standard codon usage. Our examination of the nucleotide sequence and three-frame translation of the mRNA suggest that the reading frame must be shifted 10 times, at every AGG and CCC codon, to yield a consensus COX1 protein. We suggest two possible mechanisms for these translational frameshifts: a ribosomal frameshift in which stalled ribosomes skip the first bases of these codons or specialized tRNAs recognizing non-triplet codons, AGGY and CCCCU. Regardless of the mechanism, active and efficient machinery would be required to tolerate the frameshifts predicted in Perkinsus mitochondria. To our knowledge, this is the first evidence of translational frameshifts in protist mitochondria and, by far, is the most extensive case in mitochondria.
PMCID: PMC2952869  PMID: 20507907
3.  Retrieval of Missing Spliced Leader in Dinoflagellates 
PLoS ONE  2009;4(1):e4129.
Spliced leader (SL) trans-splicing has recently been shown to be a common mRNA processing mechanism in dinoflagellates, in which a short (22-nt) sequence, DCCGUAGCCAUUUUGGCUCAAG (D = U, A, or G), is transplanted from the 5′-end of a small non-coding RNA (SL RNA) to the 5′ end of mRNA molecules. The widespread existence of the mechanism in dinoflagellates has been demonstrated by detection of this SL (DinoSL) in a wide phylogenetic range of dinoflagellates. Furthermore, the presence of DinoSL in the transcripts of highly diverse groups of nuclear-encoded genes has led us to postulate that SL trans-splicing is universal in dinoflagellate nuclear genome. However, some observations inconsistent to this postulation have been reported, exemplified by a recent article reporting apparent absence of DinoSL in the transcripts of some nuclear-encoded genes in Amphidinium carterae. Absence of SL in these gene transcripts would have important implication on gene regulation in dinoflagellates and utility of DinoSL as a universal dinoflagellate-specific primer to study dinoflagellate transcriptomics. In this study, we re-examined transcripts of these genes and found that all of them actually contained DinoSL. Therefore, results to date are consistent to our initial postulation that DinoSL occurs in all dinoflagellate nuclear-encoded mRNAs.
PMCID: PMC2606063  PMID: 19122814
4.  The Alveolate Perkinsus marinus: Biological Insights from EST Gene Discovery 
BMC Genomics  2010;11:228.
Perkinsus marinus, a protozoan parasite of the eastern oyster Crassostrea virginica, has devastated natural and farmed oyster populations along the Atlantic and Gulf coasts of the United States. It is classified as a member of the Perkinsozoa, a recently established phylum considered close to the ancestor of ciliates, dinoflagellates, and apicomplexans, and a key taxon for understanding unique adaptations (e.g. parasitism) within the Alveolata. Despite intense parasite pressure, no disease-resistant oysters have been identified and no effective therapies have been developed to date.
To gain insight into the biological basis of the parasite's virulence and pathogenesis mechanisms, and to identify genes encoding potential targets for intervention, we generated >31,000 5' expressed sequence tags (ESTs) derived from four trophozoite libraries generated from two P. marinus strains. Trimming and clustering of the sequence tags yielded 7,863 unique sequences, some of which carry a spliced leader. Similarity searches revealed that 55% of these had hits in protein sequence databases, of which 1,729 had their best hit with proteins from the chromalveolates (E-value ≤ 1e-5). Some sequences are similar to those proven to be targets for effective intervention in other protozoan parasites, and include not only proteases, antioxidant enzymes, and heat shock proteins, but also those associated with relict plastids, such as acetyl-CoA carboxylase and methyl erythrithol phosphate pathway components, and those involved in glycan assembly, protein folding/secretion, and parasite-host interactions.
Our transcriptome analysis of P. marinus, the first for any member of the Perkinsozoa, contributes new insight into its biology and taxonomic position. It provides a very informative, albeit preliminary, glimpse into the expression of genes encoding functionally relevant proteins as potential targets for chemotherapy, and evidence for the presence of a relict plastid. Further, although P. marinus sequences display significant similarity to those from both apicomplexans and dinoflagellates, the presence of trans-spliced transcripts confirms the previously established affinities with the latter. The EST analysis reported herein, together with the recently completed sequence of the P. marinus genome and the development of transfection methodology, should result in improved intervention strategies against dermo disease.
PMCID: PMC2868825  PMID: 20374649
5.  Dinoflagellate phylogeny revisited: Using ribosomal proteins to resolve deep branching dinoflagellate clades 
The alveolates are composed of three major lineages, the ciliates, dinoflagellates, and apicomplexans. Together these ‘protist’ taxa play key roles in primary production and ecology, as well as in illness of humans and other animals. The interface between the dinoflagellate and apicomplexan clades has been an area of recent discovery, blurring the distinction between these two clades. Moreover, phylogenetic analysis has yet to determine the position of basal dinoflagellate clades hence the deepest branches of the dinoflagellate tree currently remain unresolved. Large-scale mRNA sequencing was applied to 11 species of dinoflagellates, including strains of the syndinean genera Hematodinium and Amoebophrya, parasites of crustaceans and dinoflagellates, respectively, to optimize and update the dinoflagellate tree. From the transcriptome-scale data a total of 73 ribosomal protein-coding genes were selected for phylogeny. After individual gene orthology assessment, the genes were concatenated into a >15,000 amino acid alignment with 76 taxa from dinoflagellates, apicomplexans, ciliates, and the outgroup heterokonts. Overall the tree was well resolved and supported, when the data was subsampled with gblocks or constraint trees were tested with the approximately unbiased test. The deepest branches of the dinoflagellate tree can now be resolved with strong support, and provides a clearer view of the evolution of the distinctive traits of dinoflagellates.
PMCID: PMC4144664  PMID: 24135237
Dinoflagellate; Alveolate; Heterokont; Apicomplexan; Ribosomal protein
6.  Quantitative assessment of the proliferation of the protozoan parasite Perkinsus marinus using a bioluminescence assay for ATP content 
Graphical abstract
•Perkinsus marinus is a protozoan parasite that has devastated oyster populations in the USA.•Perkinsus marinus proliferation is inhibited by fluridone and atrazine.•Perkinsus marinus proliferation is inhibited by antibiotics.•The ATP-based assay can be used for the identification of leader compounds against Perkinsus and closely-related parasites.
Perkinsus marinus is a protozoan parasite that causes “Dermo” disease in the eastern oyster Crasssostrea virginica in coastal areas of the USA. Until now, intervention strategies against the parasite have found limited success, and Dermo still remains one of the main hurdles for the restoration of oyster populations. We adapted a commercial adenosine tri-phosphate (ATP) content-based assay to assess the in vitro proliferation of P. marinus in a 96-well plate format, and validated the method by measuring the effects of potential anti-proliferative compounds. The sensitivity (1.5–3.1 × 104 cells/well), linearity (R2 = 0.983), and signal stability (60 min) support the reliability of the assay for assessing cell proliferation. Validation of the assay by culturing P. marinus in the presence of increasing concentrations of triclosan showed a dose–response profile. The IC50 value obtained was higher than that reported earlier, possibly due to the use of different viability assay methods and a different P. marinus strain. The antibiotics G418 and tetracycline and the herbicide fluridone were active against P. marinus proliferation; the IC50 of chloramphenicol, ciprofloxacin, and atrazine was relatively high suggesting either off-target effects or inability to reach the targets. The validation of the ATP-based assay, together with significant advantages of the Perkinsus culture methodology (homogeneity, reproducibility, and high cell densities), underscores the value of this assay for developing high-throughput screens for the identification of novel leader compounds against Perkinsus species, and most importantly, for the closely-related apicomplexan parasites.
PMCID: PMC3862420  PMID: 24533297
Antibiotics; Herbicides; Perkinsus; Plastid; Protozoan
7.  Identification of MMV Malaria Box Inhibitors of Perkinsus marinus Using an ATP-Based Bioluminescence Assay 
PLoS ONE  2014;9(10):e111051.
“Dermo” disease caused by the protozoan parasite Perkinsus marinus (Perkinsozoa) is one of the main obstacles to the restoration of oyster populations in the USA. Perkinsus spp. are also a concern worldwide because there are limited approaches to intervention against the disease. Based on the phylogenetic affinity between the Perkinsozoa and Apicomplexa, we exposed Perkinsus trophozoites to the Medicines for Malaria Venture Malaria Box, an open access compound library comprised of 200 drug-like and 200 probe-like compounds that are highly active against the erythrocyte stage of Plasmodium falciparum. Using a final concentration of 20 µM, we found that 4 days after exposure 46% of the compounds were active against P. marinus trophozoites. Six compounds with IC50 in the µM range were used to compare the degree of susceptibility in vitro of eight P. marinus strains from the USA and five Perkinsus species from around the world. The three compounds, MMV666021, MMV665807 and MMV666102, displayed a uniform effect across Perkinsus strains and species. Both Perkinsus marinus isolates and Perkinsus spp. presented different patterns of response to the panel of compounds tested, supporting the concept of strain/species variability. Here, we expanded the range of compounds available for inhibiting Perkinsus proliferation in vitro and characterized Perkinsus phenotypes based on their resistance to six compounds. We also discuss the implications of these findings in the context of oyster management. The Perkinsus system offers the potential for investigating the mechanism of action of the compounds of interest.
PMCID: PMC4206467  PMID: 25337810
8.  The alveolate translation initiation factor 4E family reveals a custom toolkit for translational control in core dinoflagellates 
BMC Evolutionary Biology  2015;15(1):14.
Dinoflagellates are eukaryotes with unusual cell biology and appear to rely on translational rather than transcriptional control of gene expression. The eukaryotic translation initiation factor 4E (eIF4E) plays an important role in regulating gene expression because eIF4E binding to the mRNA cap is a control point for translation. eIF4E is part of an extended, eukaryote-specific family with different members having specific functions, based on studies of model organisms. Dinoflagellate eIF4E diversity could provide a mechanism for dinoflagellates to regulate gene expression in a post-transcriptional manner. Accordingly, eIF4E family members from eleven core dinoflagellate transcriptomes were surveyed to determine the diversity and phylogeny of the eIF4E family in dinoflagellates and related lineages including apicomplexans, ciliates and heterokonts.
The survey uncovered eight to fifteen (on average eleven) different eIF4E family members in each core dinoflagellate species. The eIF4E family members from heterokonts and dinoflagellates segregated into three clades, suggesting at least three eIF4E cognates were present in their common ancestor. However, these three clades are distinct from the three previously described eIF4E classes, reflecting diverse approaches to a central eukaryotic function. Heterokonts contain four clades, ciliates two and apicomplexans only a single recognizable eIF4E clade. In the core dinoflagellates, the three clades were further divided into nine sub-clades based on the phylogenetic analysis and species representation. Six of the sub-clades included at least one member from all eleven core dinoflagellate species, suggesting duplication in their shared ancestor. Conservation within sub-clades varied, suggesting different selection pressures.
Phylogenetic analysis of eIF4E in core dinoflagellates revealed complex layering of duplication and conservation when compared to other eukaryotes. Our results suggest that the diverse eIF4E family in core dinoflagellates may provide a toolkit to enable selective translation as a strategy for controlling gene expression in these enigmatic eukaryotes.
Electronic supplementary material
The online version of this article (doi:10.1186/s12862-015-0301-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4330643
Dinoflagellates; Translation initiation; eIF4E; Phylogeny; Alveolate; Heterokont
9.  Comparative Genomic Analysis of Multi-Subunit Tethering Complexes Demonstrates an Ancient Pan-Eukaryotic Complement and Sculpting in Apicomplexa 
PLoS ONE  2013;8(9):e76278.
Apicomplexa are obligate intracellular parasites that cause tremendous disease burden world-wide. They utilize a set of specialized secretory organelles in their invasive process that require delivery of components for their biogenesis and function, yet the precise mechanisms underpinning such processes remain unclear. One set of potentially important components is the multi-subunit tethering complexes (MTCs), factors increasingly implicated in all aspects of vesicle-target interactions. Prompted by the results of previous studies indicating a loss of membrane trafficking factors in Apicomplexa, we undertook a bioinformatic analysis of MTC conservation. Building on knowledge of the ancient presence of most MTC proteins, we demonstrate the near complete retention of MTCs in the newly available genomes for Guillardiatheta and Bigelowiellanatans. The latter is a key taxonomic sampling point as a basal sister taxa to the group including Apicomplexa. We also demonstrate an ancient origin of the CORVET complex subunits Vps8 and Vps3, as well as the TRAPPII subunit Tca17. Having established that the lineage leading to Apicomplexa did at one point possess the complete eukaryotic complement of MTC components, we undertook a deeper taxonomic investigation in twelve apicomplexan genomes. We observed excellent conservation of the VpsC core of the HOPS and CORVET complexes, as well as the core TRAPP subunits, but sparse conservation of TRAPPII, COG, Dsl1, and HOPS/CORVET-specific subunits. However, those subunits that we did identify appear to be expressed with similar patterns to the fully conserved MTC proteins, suggesting that they may function as minimal complexes or with analogous partners. Strikingly, we failed to identify any subunits of the exocyst complex in all twelve apicomplexan genomes, as well as the dinoflagellate Perkinsus marinus. Overall, we demonstrate reduction of MTCs in Apicomplexa and their ancestors, consistent with modification during, and possibly pre-dating, the move from free-living marine algae to deadly human parasites.
PMCID: PMC3785458  PMID: 24086721
10.  Broad genomic and transcriptional analysis reveals a highly derived genome in dinoflagellate mitochondria 
BMC Biology  2007;5:41.
Dinoflagellates comprise an ecologically significant and diverse eukaryotic phylum that is sister to the phylum containing apicomplexan endoparasites. The mitochondrial genome of apicomplexans is uniquely reduced in gene content and size, encoding only three proteins and two ribosomal RNAs (rRNAs) within a highly compacted 6 kb DNA. Dinoflagellate mitochondrial genomes have been comparatively poorly studied: limited available data suggest some similarities with apicomplexan mitochondrial genomes but an even more radical type of genomic organization. Here, we investigate structure, content and expression of dinoflagellate mitochondrial genomes.
From two dinoflagellates, Crypthecodinium cohnii and Karlodinium micrum, we generated over 42 kb of mitochondrial genomic data that indicate a reduced gene content paralleling that of mitochondrial genomes in apicomplexans, i.e., only three protein-encoding genes and at least eight conserved components of the highly fragmented large and small subunit rRNAs. Unlike in apicomplexans, dinoflagellate mitochondrial genes occur in multiple copies, often as gene fragments, and in numerous genomic contexts. Analysis of cDNAs suggests several novel aspects of dinoflagellate mitochondrial gene expression. Polycistronic transcripts were found, standard start codons are absent, and oligoadenylation occurs upstream of stop codons, resulting in the absence of termination codons. Transcripts of at least one gene, cox3, are apparently trans-spliced to generate full-length mRNAs. RNA substitutional editing, a process previously identified for mRNAs in dinoflagellate mitochondria, is also implicated in rRNA expression.
The dinoflagellate mitochondrial genome shares the same gene complement and fragmentation of rRNA genes with its apicomplexan counterpart. However, it also exhibits several unique characteristics. Most notable are the expansion of gene copy numbers and their arrangements within the genome, RNA editing, loss of stop codons, and use of trans-splicing.
PMCID: PMC2151934  PMID: 17897476
11.  Mitochondrial cob and cox1 Genes and Editing of the Corresponding mRNAs in Dinophysis acuminata from Narragansett Bay, with Special Reference to the Phylogenetic Position of the Genus Dinophysis▿  
Dinophysis acuminata cells were isolated from Narragansett Bay water samples in June 2005 using flow cytometry. Dinoflagellate-specific PCR primers were used to isolate small-subunit rRNA (18S rRNA), mitochondrial cytochrome b (cob), and cytochrome c oxidase I (cox1) genes and the encoded cDNAs. Maximum-likelihood analysis of a concatenated data set of ribosomal DNA and cDNA sequences of cob and cox1 showed that D. acuminata was sister to Gonyaulacoids, but without strong bootstrap support. The approximately unbiased test could not reject alternative positions of D. acuminata. To gain better resolution, mRNA editing of cob and cox1 was inferred for D. acuminata and 13 other dinoflagellate species. The location and type of editing as well as the distribution pattern in D. acuminata were generally similar to those in other dinoflagellates except for two edited sites that are unique to this species. Bayesian analyses of a matrix that recorded the location and type of editing, and of a matrix that included the protein sequences of COB and COX1 with the editing data yielded tree topologies similar to the three-gene tree but again failed to resolve the phylogenetic position of D. acuminata. However, the density of edited sites in the D. acuminata mitochondrial genes, consistent with phylogenetic trees, indicated that Dinophysis is a derived dinoflagellate lineage, diverging after other lineages such as Oxyrrhis, Amphidinium, and Symbiodinium. We demonstrate that dinoflagellate-specific PCR coupled with flow cytometry can be a useful tool to analyze genes and their transcripts from a natural dinoflagellate population.
PMCID: PMC2258633  PMID: 18165361
12.  The Mitochondrial Genome and Transcriptome of the Basal Dinoflagellate Hematodinium sp.: Character Evolution within the Highly Derived Mitochondrial Genomes of Dinoflagellates 
Genome Biology and Evolution  2011;4(1):59-72.
The sister phyla dinoflagellates and apicomplexans inherited a drastically reduced mitochondrial genome (mitochondrial DNA, mtDNA) containing only three protein-coding (cob, cox1, and cox3) genes and two ribosomal RNA (rRNA) genes. In apicomplexans, single copies of these genes are encoded on the smallest known mtDNA chromosome (6 kb). In dinoflagellates, however, the genome has undergone further substantial modifications, including massive genome amplification and recombination resulting in multiple copies of each gene and gene fragments linked in numerous combinations. Furthermore, protein-encoding genes have lost standard stop codons, trans-splicing of messenger RNAs (mRNAs) is required to generate complete cox3 transcripts, and extensive RNA editing recodes most genes. From taxa investigated to date, it is unclear when many of these unusual dinoflagellate mtDNA characters evolved. To address this question, we investigated the mitochondrial genome and transcriptome character states of the deep branching dinoflagellate Hematodinium sp. Genomic data show that like later-branching dinoflagellates Hematodinium sp. also contains an inflated, heavily recombined genome of multicopy genes and gene fragments. Although stop codons are also lacking for cox1 and cob, cox3 still encodes a conventional stop codon. Extensive editing of mRNAs also occurs in Hematodinium sp. The mtDNA of basal dinoflagellate Hematodinium sp. indicates that much of the mtDNA modification in dinoflagellates occurred early in this lineage, including genome amplification and recombination, and decreased use of standard stop codons. Trans-splicing, on the other hand, occurred after Hematodinium sp. diverged. Only RNA editing presents a nonlinear pattern of evolution in dinoflagellates as this process occurs in Hematodinium sp. but is absent in some later-branching taxa indicating that this process was either lost in some lineages or developed more than once during the evolution of the highly unusual dinoflagellate mtDNA.
PMCID: PMC3268668  PMID: 22113794
mitochondrion; Apicomplexa; RNA editing; organelle genome
13.  Description of Colponema vietnamica sp.n. and Acavomonas peruviana n. gen. n. sp., Two New Alveolate Phyla (Colponemidia nom. nov. and Acavomonidia nom. nov.) and Their Contributions to Reconstructing the Ancestral State of Alveolates and Eukaryotes 
PLoS ONE  2014;9(4):e95467.
The evolutionary and ecological importance of predatory flagellates are too often overlooked. This is not only a gap in our understanding of microbial diversity, but also impacts how we interpret their better-studied relatives. A prime example of these problems is found in the alveolates. All well-studied species belong to three large clades (apicomplexans, dinoflagellates, and ciliates), but the predatory colponemid flagellates are also alveolates that are rare in nature and seldom cultured, but potentially important to our understanding of alveolate evolution. Recently we reported the first cultivation and molecular analysis of several colponemid-like organisms representing two novel clades in molecular trees. Here we provide ultrastructural analysis and formal species descriptions for both new species, Colponema vietnamica n. sp. and Acavomonas peruviana n. gen. n. sp. Morphological and feeding characteristics concur with molecular data that both species are distinct members of alveolates, with Acavomonas lacking the longitudinal phagocytotic groove, a defining feature of Colponema. Based on ultrastructure and molecular phylogenies, which both provide concrete rationale for a taxonomic reclassification of Alveolata, we establish the new phyla Colponemidia nom. nov. for the genus Colponema and its close relatives, and Acavomonidia nom. nov. for the genus Acavomonas and its close relatives. The morphological data presented here suggests that colponemids are central to our understanding of early alveolate evolution, and suggest they also retain features of the common ancestor of all eukaryotes.
PMCID: PMC3989336  PMID: 24740116
14.  Red and Problematic Green Phylogenetic Signals among Thousands of Nuclear Genes from the Photosynthetic and Apicomplexa-Related Chromera velia 
Genome Biology and Evolution  2011;3:1220-1230.
The photosynthetic and basal apicomplexan Chromera velia was recently described, expanding the membership of this otherwise nonphotosynthetic group of parasite protists. Apicomplexans are alveolates with secondary plastids of red algal origin, but the evolutionary history of their nuclear genes is still actively discussed. Using deep sequencing of expressed genes, we investigated the phylogenetic affinities of a stringent filtered set of 3,151 expressed sequence tag-contigs by generating clusters with eukaryotic homologs and constructing phylogenetic trees and networks. The phylogenetic positioning of this alveolate alga was determined and sets of phyla-specific proteins extracted. Phylogenetic trees provided conflicting signals, with 444 trees grouping C. velia with the apicomplexans but 354 trees grouping C. velia with the alveolate oyster pathogen Perkinsus marinus, the latter signal being reinforced from the analysis of shared genes and overall sequence similarity. Among the 513 C. velia nuclear genes that reflect a photosynthetic ancestry and for which nuclear homologs were available both from red and green lineages, 263 indicated a red photosynthetic ancestry, whereas 250 indicated a green photosynthetic ancestry. The same 1:1 signal ratio was found among the putative 255 nuclear-encoded plastid proteins identified. This finding of red and green signals for the alveolate mirrors the result observed in the heterokont lineage and supports a common but not necessarily single origin for the plastid in heterokonts and alveolates. The inference of green endosymbiosis preceding red plastid acquisition in these lineages leads to worryingly complicated evolutionary scenarios, prompting the search for other explanations for the green phylogenetic signal and the amount of hosts involved.
PMCID: PMC3205606  PMID: 21965651
Chromera; Apicomplexa; Alveolata; chromalveolata; apicoplast; protist evolution
15.  Genetic Diversity, Morphological Uniformity and Polyketide Production in Dinoflagellates (Amphidinium, Dinoflagellata) 
PLoS ONE  2012;7(6):e38253.
Dinoflagellates are an intriguing group of eukaryotes, showing many unusual morphological and genetic features. Some groups of dinoflagellates are morphologically highly uniform, despite indications of genetic diversity. The species Amphidinium carterae is abundant and cosmopolitan in marine environments, grows easily in culture, and has therefore been used as a ‘model’ dinoflagellate in research into dinoflagellate genetics, polyketide production and photosynthesis. We have investigated the diversity of ‘cryptic’ species of Amphidinium that are morphologically similar to A. carterae, including the very similar species Amphidinium massartii, based on light and electron microscopy, two nuclear gene regions (LSU rDNA and ITS rDNA) and one mitochondrial gene region (cytochrome b). We found that six genetically distinct cryptic species (clades) exist within the species A. massartii and four within A. carterae, and that these clades differ from one another in molecular sequences at levels comparable to other dinoflagellate species, genera or even families. Using primers based on an alignment of alveolate ketosynthase sequences, we isolated partial ketosynthase genes from several Amphidinium species. We compared these genes to known dinoflagellate ketosynthase genes and investigated the evolution and diversity of the strains of Amphidinium that produce them.
PMCID: PMC3366924  PMID: 22675531
16.  Analysis of EST data of the marine protist Oxyrrhis marina, an emerging model for alveolate biology and evolution 
BMC Genomics  2014;15:122.
The alveolates include a large number of important lineages of protists and algae, among which are three major eukaryotic groups: ciliates, apicomplexans and dinoflagellates. Collectively alveolates are present in virtually every environment and include a vast diversity of cell shapes, molecular and cellular features and feeding modes including lifestyles such as phototrophy, phagotrophy/predation and intracellular parasitism, in addition to a variety of symbiotic associations. Oxyrrhis marina is a well-known model for heterotrophic protist biology, and is now emerging as a useful organism to explore the many changes that occurred during the origin and diversification of dinoflagellates by virtue of its phylogenetic position at the base of the dinoflagellate tree.
We have generated and analysed expressed sequence tag (EST) sequences from the alveolate Oxyrrhis marina in order to shed light on the evolution of a number of dinoflagellate characteristics, especially regarding the emergence of highly unusual genomic features. We found that O. marina harbours extensive gene redundancy, indicating high rates of gene duplication and transcription from multiple genomic loci. In addition, we observed a correlation between expression level and copy number in several genes, suggesting that copy number may contribute to determining transcript levels for some genes. Finally, we analyze the genes and predicted products of the recently discovered Dinoflagellate Viral Nuclear Protein, and several cases of horizontally acquired genes.
The dataset presented here has proven very valuable for studying this important group of protists. Our analysis indicates that gene redundancy is a pervasive feature of dinoflagellate genomes, thus the mechanisms involved in its generation must have arisen early in the evolution of the group.
PMCID: PMC3942190  PMID: 24512041
Dinoflagellates; Alveolates; Chromatin; Genome; Oxyrrhis
17.  Evolutionary Acquisition and Loss of Saxitoxin Biosynthesis in Dinoflagellates: the Second “Core” Gene, sxtG 
Saxitoxin and its derivatives are potent neurotoxins produced by several cyanobacteria and dinoflagellate species. SxtA is the initial enzyme in the biosynthesis of saxitoxin. The dinoflagellate full mRNA and partial genomic sequences have previously been characterized, and it appears that sxtA originated in dinoflagellates through a horizontal gene transfer from a bacterium. So far, little is known about the remaining genes involved in this pathway in dinoflagellates. Here we characterize sxtG, an amidinotransferase enzyme gene that putatively encodes the second step in saxitoxin biosynthesis. In this study, the entire sxtG transcripts from Alexandrium fundyense CCMP1719 and Alexandrium minutum CCMP113 were amplified and sequenced. The transcripts contained typical dinoflagellate spliced leader sequences and eukaryotic poly(A) tails. In addition, partial sxtG transcript fragments were amplified from four additional Alexandrium species and Gymnodinium catenatum. The phylogenetic inference of dinoflagellate sxtG, congruent with sxtA, revealed a bacterial origin. However, it is not known if sxtG was acquired independently of sxtA. Amplification and sequencing of the corresponding genomic sxtG region revealed noncanonical introns. These introns show a high interspecies and low intraspecies variance, suggesting multiple independent acquisitions and losses. Unlike sxtA, sxtG was also amplified from Alexandrium species not known to synthesize saxitoxin. However, amplification was not observed for 22 non-saxitoxin-producing dinoflagellate species other than those of the genus Alexandrium or G. catenatum. This result strengthens our hypothesis that saxitoxin synthesis has been secondarily lost in conjunction with sxtA for some descendant species.
PMCID: PMC3623241  PMID: 23335767
18.  Highly Divergent Mitochondrial ATP Synthase Complexes in Tetrahymena thermophila 
PLoS Biology  2010;8(7):e1000418.
Tetrahymena ATP synthase, an evolutionarily divergent protein complex, has a very unusual structure and protein composition including a unique Fo subunit a and at least 13 proteins with no orthologs outside of the ciliate lineage.
The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F1 sector catalyzes ATP synthesis, whereas the Fo sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F1 and Fo sectors are highly conserved across prokaryotes and eukaryotes. Therefore, it was a surprise that genes encoding the a and b subunits as well as other components of the Fo sector were undetectable in the sequenced genomes of a variety of apicomplexan parasites. While the parasitic existence of these organisms could explain the apparent incomplete nature of ATP synthase in Apicomplexa, genes for these essential components were absent even in Tetrahymena thermophila, a free-living ciliate belonging to a sister clade of Apicomplexa, which demonstrates robust oxidative phosphorylation. This observation raises the possibility that the entire clade of Alveolata may have invented novel means to operate ATP synthase complexes. To assess this remarkable possibility, we have carried out an investigation of the ATP synthase from T. thermophila. Blue native polyacrylamide gel electrophoresis (BN-PAGE) revealed the ATP synthase to be present as a large complex. Structural study based on single particle electron microscopy analysis suggested the complex to be a dimer with several unique structures including an unusually large domain on the intermembrane side of the ATP synthase and novel domains flanking the c subunit rings. The two monomers were in a parallel configuration rather than the angled configuration previously observed in other organisms. Proteomic analyses of well-resolved ATP synthase complexes from 2-D BN/BN-PAGE identified orthologs of seven canonical ATP synthase subunits, and at least 13 novel proteins that constitute subunits apparently limited to the ciliate lineage. A mitochondrially encoded protein, Ymf66, with predicted eight transmembrane domains could be a substitute for the subunit a of the Fo sector. The absence of genes encoding orthologs of the novel subunits even in apicomplexans suggests that the Tetrahymena ATP synthase, despite core similarities, is a unique enzyme exhibiting dramatic differences compared to the conventional complexes found in metazoan, fungal, and plant mitochondria, as well as in prokaryotes. These findings have significant implications for the origins and evolution of a central player in bioenergetics.
Author Summary
Synthesis of ATP, the currency of the cellular energy economy, is carried out by a rotary nano-motor, the ATP synthase complex, which uses proton flow to drive the rotation of protein subunits so as to produce ATP. There are two main components in mitochondrial F-type ATP synthase complexes, each made up of a number of different proteins: F1 has the catalytic sites for ATP synthesis, and Fo forms channels for proton movement and provides a bearing and stator to contain the rotary action of the motor. The two parts of the complex have to interact with each other, and critical protein subunits of the enzyme are conserved from bacteria to higher eukaryotes. We were surprised that a group of unicellular organisms called alveolates (including ciliates, apicomplexa, and dinoflagellates) seemed to lack two critical proteins of the Fo component. We have isolated intact ATP synthase complexes from the ciliate Tetrahymena thermophila and examined their structure by electron microscopy and their protein composition by mass spectrometry. We found that the ATP synthase complex of this organism is quite different, both in its overall structure and in many of the associated protein subunits, from the ATP synthase in other organisms. At least 13 novel proteins are present within this complex that have no orthologs in any organism outside of the ciliates. Our results suggest significant divergence of a critical bioenergetic player within the alveolate group.
PMCID: PMC2903591  PMID: 20644710
19.  Real-Time PCR for Detection and Quantification of the Protistan Parasite Perkinsus marinus in Environmental Waters†  
Applied and Environmental Microbiology  2004;70(11):6611-6618.
The protistan parasite Perkinsus marinus is a severe pathogen of the oyster Crassostrea virginica along the east coast of the United States. Very few data have been collected, however, on the abundance of the parasite in environmental waters, limiting our understanding of P. marinus transmission dynamics. Real-time PCR assays with SybrGreen I as a label for detection were developed in this study for quantification of P. marinus in environmental waters with P. marinus species-specific primers and of Perkinsus spp. with Perkinsus genus-specific primers. Detection of DNA concentrations as low as the equivalent of 3.3 × 10−2 cell per 10-μl reaction mixture was obtained by targeting the multicopy internal transcribed spacer region of the genome. To obtain reliable target quantification from environmental water samples, removal of PCR inhibitors and efficient DNA recovery were two major concerns. A DNA extraction kit designed for tissues and another designed for stool samples were tested on environmental and artificial seawater (ASW) samples spiked with P. marinus cultured cells. The stool kit was significantly more efficient than the tissue kit at removing inhibitors from environmental water samples. With the stool kit, no significant difference in the quantified target concentrations was observed between the environmental and ASW samples. However, with the spiked ASW samples, the tissue kit demonstrated more efficient DNA recovery. Finally, by performing three elutions of DNA from the spin columns, which were combined prior to target quantification, variability of DNA recovery from different samples was minimized and more reliable real-time PCR quantification was accomplished.
PMCID: PMC525192  PMID: 15528525
20.  Diverse Bacterial PKS Sequences Derived From Okadaic Acid-Producing Dinoflagellates 
Marine Drugs  2008;6(2):164-179.
Okadaic acid (OA) and the related dinophysistoxins are isolated from dinoflagellates of the genus Prorocentrum and Dinophysis. Bacteria of the Roseobacter group have been associated with okadaic acid producing dinoflagellates and have been previously implicated in OA production. Analysis of 16S rRNA libraries reveals that Roseobacter are the most abundant bacteria associated with OA producing dinoflagellates of the genus Prorocentrum and are not found in association with non-toxic dinoflagellates. While some polyketide synthase (PKS) genes form a highly supported Prorocentrum clade, most appear to be bacterial, but unrelated to Roseobacter or Alpha-Proteobacterial PKSs or those derived from other Alveolates Karenia brevis or Crytosporidium parvum.
PMCID: PMC2525486  PMID: 18728765
okadaic acid; polyketide; polyketide synthase; biosynthesis; Roseobacter
21.  Evaluating the Ribosomal Internal Transcribed Spacer (ITS) as a Candidate Dinoflagellate Barcode Marker 
PLoS ONE  2012;7(8):e42780.
DNA barcoding offers an efficient way to determine species identification and to measure biodiversity. For dinoflagellates, an ancient alveolate group of about 2000 described extant species, DNA barcoding studies have revealed large amounts of unrecognized species diversity, most of which is not represented in culture collections. To date, two mitochondrial gene markers, Cytochrome Oxidase I (COI) and Cytochrome b oxidase (COB), have been used to assess DNA barcoding in dinoflagellates, and both failed to amplify all taxa and suffered from low resolution. Nevertheless, both genes yielded many examples of morphospecies showing cryptic speciation and morphologically distinct named species being genetically similar, highlighting the need for a common marker. For example, a large number of cultured Symbiodinium strains have neither taxonomic identification, nor a common measure of diversity that can be used to compare this genus to other dinoflagellates.
Methodology/Principal Findings
The purpose of this study was to evaluate the Internal Transcribed Spacer units 1 and 2 (ITS) of the rDNA operon, as a high resolution marker for distinguishing species dinoflagellates in culture. In our study, from 78 different species, the ITS barcode clearly differentiated species from genera and could identify 96% of strains to a known species or sub-genus grouping. 8.3% showed evidence of being cryptic species. A quarter of strains identified had no previous species identification. The greatest levels of hidden biodiversity came from Scrippsiella and the Pfiesteriaceae family, whilst Heterocapsa strains showed a high level of mismatch to their given species name.
The ITS marker was successful in confirming species, revealing hidden diversity in culture collections. This marker, however, may have limited use for environmental barcoding due to paralogues, the potential for unidentifiable chimaeras and priming across taxa. In these cases ITS would serve well in combination with other markers or for specific taxon studies.
PMCID: PMC3420951  PMID: 22916158
22.  Chromera velia, Endosymbioses and the Rhodoplex Hypothesis—Plastid Evolution in Cryptophytes, Alveolates, Stramenopiles, and Haptophytes (CASH Lineages)  
Genome Biology and Evolution  2014;6(3):666-684.
The discovery of Chromera velia, a free-living photosynthetic relative of apicomplexan pathogens, has provided an unexpected opportunity to study the algal ancestry of malaria parasites. In this work, we compared the molecular footprints of a eukaryote-to-eukaryote endosymbiosis in C. velia to their equivalents in peridinin-containing dinoflagellates (PCD) to reevaluate recent claims in favor of a common ancestry of their plastids. To this end, we established the draft genome and a set of full-length cDNA sequences from C. velia via next-generation sequencing. We documented the presence of a single coxI gene in the mitochondrial genome, which thus represents the genetically most reduced aerobic organelle identified so far, but focused our analyses on five “lucky genes” of the Calvin cycle. These were selected because of their known support for a common origin of complex plastids from cryptophytes, alveolates (represented by PCDs), stramenopiles, and haptophytes (CASH) via a single secondary endosymbiosis with a red alga. As expected, our broadly sampled phylogenies of the nuclear-encoded Calvin cycle markers support a rhodophycean origin for the complex plastid of Chromera. However, they also suggest an independent origin of apicomplexan and dinophycean (PCD) plastids via two eukaryote-to-eukaryote endosymbioses. Although at odds with the current view of a common photosynthetic ancestry for alveolates, this conclusion is nonetheless in line with the deviant plastome architecture in dinoflagellates and the morphological paradox of four versus three plastid membranes in the respective lineages. Further support for independent endosymbioses is provided by analysis of five additional markers, four of them involved in the plastid protein import machinery. Finally, we introduce the “rhodoplex hypothesis” as a convenient way to designate evolutionary scenarios where CASH plastids are ultimately the product of a single secondary endosymbiosis with a red alga but were subsequently horizontally spread via higher-order eukaryote-to-eukaryote endosymbioses.
PMCID: PMC3971594  PMID: 24572015
next-generation sequencing; eukaryote-to-eukaryote endosymbioses; horizontal and endosymbiotic gene transfer; chromalveolate hypothesis; long-branch attraction artifacts
23.  Molecular phylogeny of ocelloid-bearing dinoflagellates (Warnowiaceae) as inferred from SSU and LSU rDNA sequences 
Dinoflagellates represent a major lineage of unicellular eukaryotes with unparalleled diversity and complexity in morphological features. The monophyly of dinoflagellates has been convincingly demonstrated, but the interrelationships among dinoflagellate lineages still remain largely unresolved. Warnowiid dinoflagellates are among the most remarkable eukaryotes known because of their possession of highly elaborate ultrastructural systems: pistons, nematocysts, and ocelloids. Complex organelles like these are evolutionary innovations found only in a few athecate dinoflagellates. Moreover, the taxonomy of warnowiids is extremely confusing and inferences about the evolutionary history of this lineage are mired by the absence of molecular phylogenetic data from any member of the group. In this study, we provide the first molecular phylogenetic data for warnowiids and couple them with a review of warnowiid morphological features in order to formulate a hypothetical framework for understanding character evolution within the group. These data also enabled us to evaluate the evolutionary relationship(s) between warnowiids and the other group of dinoflagellates with complex organelles: polykrikoids.
Molecular phylogenetic analyses of SSU and LSU rDNA sequences demonstrated that warnowiids form a well-supported clade that falls within the more inclusive Gymnodinium sensu stricto clade. These data also confirmed that polykrikoids are members of the Gymnodinium sensu stricto clade as well; however, a specific sister relationship between the warnowiid clade and the polykrikoid clade was unresolved in all of our analyses. Nonetheless, the new DNA sequences from different isolates of warnowiids provided organismal anchors for several previously unidentified sequences derived from environmental DNA surveys of marine biodiversity.
Comparative morphological data and molecular phylogenetic data demonstrate that the polykrikoid and the warnowiid clade are closely related to each other, but the precise branching order within the Gymnodinium sensu stricto clade remains unresolved. We regard the ocelloid as the best synapomorphy for warnowiids and infer that the most recent common ancestor of polykrikoids and warnowiids possessed both nematocysts and photosynthetic plastids that were subsequently lost during the early evolution of warnowiids. Our summary of species and genus concepts in warnowiids demonstrate that the systematics of this poorly understood group is highly problematic and a comprehensive revision is needed.
PMCID: PMC2694157  PMID: 19467154
24.  Dinoflagellate Spliced Leader RNA Genes Display a Variety of Sequences and Genomic Arrangements 
Molecular Biology and Evolution  2009;26(8):1757-1771.
Spliced leader (SL) trans-splicing is a common mRNA processing mechanism in dinoflagellates, in which a 22-nt sequence is transferred from the 5′-end of a small noncoding RNA, the SL RNA, to the 5′-end of mRNA molecules. Although the SL RNA gene was shown initially to be organized as tandem repeats with transcripts of 50–60 nt, shorter than most of their counterparts in other organisms, other gene organizations and transcript lengths were reported subsequently. To address the evolutionary gradient of gene organization complexity, we thoroughly examined transcript and gene organization of the SL RNA in a phylogenetically and ecologically diverse group of dinoflagellates representing four Orders. All these dinoflagellates possessed SL RNA transcripts of 50–60 nt, although in one species additional transcripts of up to 92 nt were also detected. At the genomic level, various combinations of SL RNA and 5S rRNA tandem gene arrays, including SL RNA–only, 5S rRNA–only, and mixed SL RNA–5S rRNA (SL–5S) clusters, were amplified by polymerase chain reaction for six dinoflagellates, containing intergenic spacers ranging from 88 bp to over 1.2 kb. Of these species, no SL–5S cluster was detected in Prorocentrum minimum, and only Karenia brevis showed the U6 small nuclear RNA gene associated with these mixed arrays. The 5S rRNA–only array was also found in three dinoflagellates, along with two SL–5S-adjacent arrangements found in two other species that could represent junctions. Two species contained multimeric SL exon repeats with no associated intron. These results suggest that 1) both the SL RNA tandem repeat and the SL–5S cluster genomic organizations are an “ancient” and widespread feature within the phylum of dinoflagellates and 2) rampant genomic duplication and recombination are ongoing independently in each dinoflagellate lineage, giving rise to the highly complex and diversified genomic arrangements of the SL RNA gene, while conserving the length and structure of the functional SL RNA.
PMCID: PMC2734150  PMID: 19387009
Dinoflagellate; SL RNA; complex genomic arrangement
25.  Humanized HLA-DR4 Mice Fed with the Protozoan Pathogen of Oysters Perkinsus Marinus (Dermo) Do Not Develop Noticeable Pathology but Elicit Systemic Immunity 
PLoS ONE  2014;9(1):e87435.
Perkinsus marinus (Phylum Perkinsozoa) is a marine protozoan parasite responsible for “Dermo” disease in oysters, which has caused extensive damage to the shellfish industry and estuarine environment. The infection prevalence has been estimated in some areas to be as high as 100%, often causing death of infected oysters within 1–2 years post-infection. Human consumption of the parasites via infected oysters is thus likely to occur, but to our knowledge the effect of oral consumption of P. marinus has not been investigated in humans or other mammals. To address the question we used humanized mice expressing HLA-DR4 molecules and lacking expression of mouse MHC-class II molecules (DR4.EA0) in such a way that CD4 T cell responses are solely restricted by the human HLA-DR4 molecule. The DR4.EA0 mice did not develop diarrhea or any detectable pathology in the gastrointestinal tract or lungs following single or repeated feedings with live P. marinus parasites. Furthermore, lymphocyte populations in the gut associated lymphoid tissue and spleen were unaltered in the parasite-fed mice ruling out local or systemic inflammation. Notably, naïve DR4.EA0 mice had antibodies (IgM and IgG) reacting against P. marinus parasites whereas parasite specific T cell responses were undetectable. Feeding with P. marinus boosted the antibody responses and stimulated specific cellular (IFNγ) immunity to the oyster parasite. Our data indicate the ability of P. marinus parasites to induce systemic immunity in DR4.EA0 mice without causing noticeable pathology, and support rationale grounds for using genetically engineered P. marinus as a new oral vaccine platform to induce systemic immunity against infectious agents.
PMCID: PMC3909113  PMID: 24498105

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