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

The Cyst-Theca Relationship Of The Dinoflagellate Cyst Trinovantedinium Pallidifulvum, With Erection Of Protoperidinium Lousianensis Sp Nov And Their Phylogenetic Position Within The Conica Group

We establish the cyst-theca relationship of the dinoflagellate cyst species Trinovantedinium pallidifulvum Matsuoka 1987 based on germination experiments of specimens isolated from the Gulf of Mexico. We show that the motile stage is a new species, designated as Protoperidinium louisianensis. We also determine its phylogenetic position based on single-cell polymerase chain reaction (PCR) of a single cell germinated from the Gulf of Mexico cysts. To further refine the phylogeny, we determined the large subunit (LSU) sequence through single-cell PCR of the cyst Selenopemphix undulata isolated from Brentwood Bay (Saanich Inlet, BC, Canada). The phylogeny shows that P. louisianensis is closest to P. shanghaiense, the motile stage of T. applanatum, and is consistent with the monophyly of the genus Trinovantedinium. Selenopemphix undulata belongs to a different clade than Selenopemphix quanta (alleged cyst of P. conicum), suggesting that the genus Selenopemphix is polyphyletic. Trinovantedinium pallidifulvum is widely distributed with occurrences in the Gulf of Mexico, the North Atlantic, the northeast Pacific and southeast Asia. In addition, we illustrate the two other extant species, Trinovantedinium applanatum and Trinovantedinium variabile, and two morphotypes of Trinovantedinium. Geochemical analyses of the cyst wall of T. pallidifulvum indicate the presence of amide groups in agreement with other heterotrophic dinoflagellate species, although the cyst wall of T. pallidifulvum also includes some unique features

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

Localization of Pyrophosphatase and V-ATPase in Chlamydomonas reinhardtii

Microsomal membranes of Chlamydomonas reinhardtii possess PPase and V‐ATPase activities. By immunogold labelling we have shown that H+‐pyrophosphatase (PPase) is localized to membranes of lytic and contractile vacuoles of Chlamydomonas, in which the density of antigen in the latter is much higher. In addition, PPase is conspicuously present in trans cisternae and transpole elements of the Colgi apparatus. Such a distribution for PPase has hitherto not been reported. A positive in situ identification for PPase at the plasma membrane, including the flagellar membrane, was also made, and has also been confirmed by Western blotting and activity measurements on isolated plasma membranes. V‐ATPase antisera which cross react with polypeptides of this transport complex from maize roots failed to recognize anything in Western blots of Chlamydomonas microsomal membranes. Thus immunogold labelling for V‐ATPase was not possible with Chlamydomonas. On the other hand, surfaces of contractile vacuole membranes as revealed by deepetching were covered by conspicuous 9 − 11.5 nm diameter smooth particles which had a central hole. These were very similar to those previously identified by Heuser et al., (1993) as the V,‐head of V‐ATPase in Dictyostelium contractile vacuoles. Another type of membrane image, designated “intermediate‐sized vesicle”, was found associated with the contractile vacuole. It was characterized by densely‐packed 6 − 7.5nm diameter polygonal particles, which upon rotation analysis showed both 5‐ and 6‐fold symmetries, also with a central hole. These particles are interpreted as representing either PPase complexes or the V0 body of the V‐ATPase in etched fractured membrane surfaces. We have incorporated these findings into a model of contractile vacuole function