Environmental Barcoding Reveals Massive Dinoflagellate Diversity in Marine Environments

Rowena F. Stern is with University of British Columbia, Ales Horak is with University of British Columbia, Rose L. Andrew is with University of British Columbia, Mary-Alice Coffroth is with State University of New York at Buffalo, Robert A. Andersen is with the Bigelow Laboratory for Ocean Sciences, Frithjof C. Küpper is with the Scottish Marine Institute, Ian Jameson is with CSIRO Marine and Atmospheric Research, Mona Hoppenrath is with the German Center for Marine Biodiversity Research, Benoît Véron is with University of Caen Lower Normandy and the National Institute for Environmental Studies, Fumai Kasai is with the National Institute for Environmental Studies, Jerry Brand is with UT Austin, Erick R. James is with University of British Columbia, Patrick J. Keeling is with University of British Columbia.Background -- Dinoflagellates are an ecologically important group of protists with important functions as primary producers, coral symbionts and in toxic red tides. Although widely studied, the natural diversity of dinoflagellates is not well known. DNA barcoding has been utilized successfully for many protist groups. We used this approach to systematically sample known “species”, as a reference to measure the natural diversity in three marine environments. Methodology/Principal Findings -- In this study, we assembled a large cytochrome c oxidase 1 (COI) barcode database from 8 public algal culture collections plus 3 private collections worldwide resulting in 336 individual barcodes linked to specific cultures. We demonstrate that COI can identify to the species level in 15 dinoflagellate genera, generally in agreement with existing species names. Exceptions were found in species belonging to genera that were generally already known to be taxonomically challenging, such as Alexandrium or Symbiodinium. Using this barcode database as a baseline for cultured dinoflagellate diversity, we investigated the natural diversity in three diverse marine environments (Northeast Pacific, Northwest Atlantic, and Caribbean), including an evaluation of single-cell barcoding to identify uncultivated groups. From all three environments, the great majority of barcodes were not represented by any known cultured dinoflagellate, and we also observed an explosion in the diversity of genera that previously contained a modest number of known species, belonging to Kareniaceae. In total, 91.5% of non-identical environmental barcodes represent distinct species, but only 51 out of 603 unique environmental barcodes could be linked to cultured species using a conservative cut-off based on distances between cultured species. Conclusions/Significance -- COI barcoding was successful in identifying species from 70% of cultured genera. When applied to environmental samples, it revealed a massive amount of natural diversity in dinoflagellates. This highlights the extent to which we underestimate microbial diversity in the environment.This project was funded by Genome Canada and the Canadian Barcode of Life Network. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Biological Sciences, School o

Genetic Diversity, Morphological Uniformity and Polyketide Production in Dinoflagellates (Amphidinium, Dinoflagellata)

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

Identifying and Characterizing Alternative Molecular Markers for the Symbiotic and Free-Living Dinoflagellate Genus Symbiodinium

Dinoflagellates in the genus Symbiodinium are best known as endosymbionts of corals and other invertebrate as well as protist hosts, but also exist free-living in coastal environments. Despite their importance in marine ecosystems, less than 10 loci have been used to explore phylogenetic relationships in this group, and only the multi-copy nuclear ribosomal Internal Transcribed Spacer (ITS) regions 1 and 2 have been used to characterize fine-scale genetic diversity within the nine clades (A–I) that comprise the genus. Here, we describe a three-step molecular approach focused on 1) identifying new candidate genes for phylogenetic analysis of Symbiodinium spp., 2) characterizing the phylogenetic relationship of these candidate genes from DNA samples spanning eight Symbiodinium clades (A–H), and 3) conducting in-depth phylogenetic analyses of candidate genes displaying genetic divergences equal or higher than those within the ITS-2 of Symbiodinium clade C. To this end, we used bioinformatics tools and reciprocal comparisons to identify homologous genes from 55,551 cDNA sequences representing two Symbiodinium and six additional dinoflagellate EST libraries. Of the 84 candidate genes identified, 7 Symbiodinium genes (elf2, coI, coIII, cob, calmodulin, rad24, and actin) were characterized by sequencing 23 DNA samples spanning eight Symbiodinium clades (A–H). Four genes displaying higher rates of genetic divergences than ITS-2 within clade C were selected for in-depth phylogenetic analyses, which revealed that calmodulin has limited taxonomic utility but that coI, rad24, and actin behave predictably with respect to Symbiodinium lineage C and are potential candidates as new markers for this group. The approach for targeting candidate genes described here can serve as a model for future studies aimed at identifying and testing new phylogenetically informative genes for taxa where transcriptomic and genomics data are available

Marine benthic dinoflagellates - unveiling their worldwide biodiversity

This publication presents the first summary of our knowledge of benthic dinoflagellate species. Dinoflagellates are important primary producers and symbionts, but, at the same time, also consumers and parasites. Species compositions of benthic habitats are quite distinct from those of planktonic habitats. Less than 10% of the approximately 2000 described extant dinoflagellate species appear to be benthic. They occur in different types of habitats (chapter II) and their morphology, their behavior, and some of their life cycles (chapter VI) seem to be well adapted to the benthic lifestyle. Information on their geographic distribution is still very limited and is compiled herein (chapter V). The study of harmful benthic dinoflagellates started in the late 1970s when it was suspected that a benthic species, later named Gambierdiscus toxicus, was responsible for ciguatera fish poisoning, a type of human poisoning linked to the consumption of certain species of tropical reef fish. As the number of ciguatera fish poisoning incidents increases, and the distribution of toxin producing benthic taxa seems to be expanding, detailed understanding of the species diversity and the ability to accurately identify them is becoming increasingly important (chapter VII). Dinoflagellate classification is currently undergoing changes and far from being settled, as new species and genera are discovered and systematic entities are rearranged. Many benthic dinoflagellate genera have unusual morphologies and appear to be only remotely related to known planktonic taxa, so that molecular phylogenetic analyses frequently show little statistical support for any relationship (chapter IV). Benthic species display unique thecal plate arrangements compared to planktonic species, e.g. Adenoides, Amphidiniella, Cabra, Planodinium, Sabulodinium, Rhinodinium (chapter III). Therefore, no classification on higher rank levels (e.g. family, order) was used throughout this book. Genera (and species within a genus) are presented in alphabetical order. The book presents the first comprehensive identification help for benthic dinoflagellates. At the same time it aims to lend support in order to improve monitoring efforts worldwide. About 190 species in 45 genera are presented in detail, illustrated with more than 200 color images, approximately 150 scanning electron micrographs, and more than 250 drawings

Characterization of Gambierdiscus lapillus sp. nov. (Gonyaulacales, Dinophyceae): a new toxic dinoflagellate from the Great Barrier Reef (Australia)

© 2016 Phycological Society of America Gambierdiscus is a genus of benthic dinoflagellates found worldwide. Some species produce neurotoxins (maitotoxins and ciguatoxins) that bioaccumulate and cause ciguatera fish poisoning (CFP), a potentially fatal food-borne illness that is common worldwide in tropical regions. The investigation of toxigenic species of Gambierdiscus in CFP endemic regions in Australia is necessary as a first step to determine which species of Gambierdiscus are related to CFP cases occurring in this region. In this study, we characterized five strains of Gambierdiscus collected from Heron Island, Australia, a region in which ciguatera is endemic. Clonal cultures were assessed using (i) light microscopy; (ii) scanning electron microscopy; (iii) DNA sequencing based on the nuclear encoded ribosomal 18S and D8-D10 28S regions; (iv) toxicity via mouse bioassay; and (v) toxin profile as determined by Liquid Chromatography-Mass Spectrometry. Both the morphological and phylogenetic data indicated that these strains represent a new species of Gambierdiscus, G. lapillus sp. nov. (plate formula Po, 3′, 0a, 7″, 6c, 7-8s, 5‴, 0p, 2″″ and distinctive by size and hatchet-shaped 2′ plate). Culture extracts were found to be toxic using the mouse bioassay. Using chemical analysis, it was determined that they did not contain maitotoxin (MTX1) or known algal-derived ciguatoxin analogs (CTX3B, 3C, CTX4A, 4B), but that they contained putative MTX3, and likely other unknown compounds

Taxonomy and phylogeny of the benthic Prorocentrum species (Dinophyceae)-A proposal and review

Based on a literature review and new morphological and molecular phylogenetic data, a summary of all described benthic Prorocentrum species is presented. Short descriptions of the 29 species are provided including information and illustrations of platelet pattern. Increasing morphological data of the same taxon from different sites has revealed unexpected morphological variability. The variability of characters used for taxonomy is discussed. Many of these may be more variable than previously thought or phenotypically plastic. We conclude that the following features are constant within a species: (1) asymmetry or symmetry of the theca in combination with (2) the shape of the periflagellar area visible on the right thecal plate, (a) arc-shaped, (b) wide or simply V-shaped, (c) narrow and deep V-shaped, and (d) linear. At the same time, we are now beginning to understand that the cell shape of some species is more variable than thought and not in others. The stability of pore patterns is not known to date nor is it known whether the presence of a starch sheath is a reliable taxonomic character. The morphology of the periflagellar area might be a very useful character. The unstable terminology to describe similar or identical structures has made it difficult to interpret the literature on these features. Therefore a unified revised terminology for morphological features, cell orientation, and platelet numbering are proposed. This review highlights uncertainty in species boundaries of several potentially toxin producing species. This includes the species Prorocentrum hoffmannianum, Prorocentrum belizeanum, Prorocentrum maculosum, and Prorocentrum faustiae. Some species are reported to have variable toxicity, e.g. P. belizeanum and Prorocentrum rhathymum. It is clear that several species complexes, containing genetically related cryptic species, are present. The species complexes proposed in the past and in this paper reflect the difficulties in distinguishing these species, which may be highly morphologically similar to one another, sometimes despite genetic differences. Additionally, the growing molecular data set has allowed us to evaluate current species concepts based on novel molecular genetic information. As some Prorocentrum species are potentially toxic and are included in monitoring programs, it is important to evaluate species boundaries as currently understood, and to determine our current knowledge gaps regarding the relationship between species and strain identification and toxin production. © 2013 Elsevier B.V