Mona Hoppenrath

The revised classification of eukaryotes.

This revision of the classification of eukaryotes, which updates that of Adl et al. [J. Eukaryot. Microbiol. 52 (2005) 399], retains an emphasis on the protists and incorporates changes since 2005 that have resolved nodes and branches in phylogenetic trees. Whereas the previous revision was successful in re‐introducing name stability to the classification, this revision provides a classification for lineages that were then still unresolved. The supergroups have withstood phylogenetic hypothesis testing with some modifications, but despite some progress, problematic nodes at the base of the eukaryotic tree still remain to be statistically resolved. Looking forward, subsequent transformations to our understanding of the diversity of life will be from the discovery of novel lineages in previously under‐sampled areas and from environmental genomic information.

Environmental Barcoding Reveals Massive Dinoflagellate Diversity in Marine Environments

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.

Dinoflagellate Phylogeny as Inferred from Heat Shock Protein 90 and Ribosomal Gene Sequences

Background Interrelationships among dinoflagellates in molecular phylogenies are largely unresolved, especially in the deepest branches. Ribosomal DNA (rDNA) sequences provide phylogenetic signals only at the tips of the dinoflagellate tree. Two reasons for the poor resolution of deep dinoflagellate relationships using rDNA sequences are (1) most sites are relatively conserved and (2) there are different evolutionary rates among sites in different lineages. Therefore, alternative molecular markers are required to address the deeper phylogenetic relationships among dinoflagellates. Preliminary evidence indicates that the heat shock protein 90 gene (Hsp90) will provide an informative marker, mainly because this gene is relatively long and appears to have relatively uniform rates of evolution in different lineages. Methodology/Principal Findings We more than doubled the previous dataset of Hsp90 sequences from dinoflagellates by generating additional sequences from 17 different species, representing seven different orders. In order to concatenate the Hsp90 data with rDNA sequences, we supplemented the Hsp90 sequences with three new SSU rDNA sequences and five new LSU rDNA sequences. The new Hsp90 sequences were generated, in part, from four additional heterotrophic dinoflagellates and the type species for six different genera. Molecular phylogenetic analyses resulted in a paraphyletic assemblage near the base of the dinoflagellate tree consisting of only athecate species. However, Noctiluca was never part of this assemblage and branched in a position that was nested within other lineages of dinokaryotes. The phylogenetic trees inferred from Hsp90 sequences were consistent with trees inferred from rDNA sequences in that the backbone of the dinoflagellate clade was largely unresolved. Conclusions/Significance The sequence conservation in both Hsp90 and rDNA sequences and the poor resolution of the deepest nodes suggests that dinoflagellates reflect an explosive radiation in morphological diversity in their recent evolutionary past. Nonetheless, the more comprehensive analysis of Hsp90 sequences enabled us to infer phylogenetic interrelationships of dinoflagellates more rigorously. For instance, the phylogenetic position of Noctiluca, which possesses several unusual features, was incongruent with previous phylogenetic studies. Therefore, the generation of additional dinoflagellate Hsp90 sequences is expected to refine the stem group of athecate species observed here and contribute to future multi-gene analyses of dinoflagellate interrelationships.

Dinoflagellate, Euglenid, or Cercomonad? The Ultrastructure and Molecular Phylogenetic Position of Protaspis grandis n. sp.

ABSTRACT. Protaspis is an enigmatic genus of marine phagotrophic biflagellates that have been tentatively classified with several different groups of eukaryotes, including dinoflagellates, euglenids, and cercomonads. This uncertainty led us to investigate the phylogenetic position of Protaspis grandis n. sp. with ultrastructural and small subunit (SSU) rDNA sequence data. Our results demonstrated that the cells were dorsoventrally flattened, shaped like elongated ovals with parallel lateral sides, 32.5–55.0 μm long and 20.0–35.0 μm wide. Moreover, two heterodynamic flagella emerged through funnels that were positioned subapically, each within a depression and separated by a distinctive protrusion. A complex multilayered wall surrounded the cell. Like dinoflagellates and euglenids, the nucleus contained permanently condensed chromosomes and a large nucleolus throughout the cell cycle. Pseudopodia containing numerous mitochondria with tubular cristae emerged from a ventral furrow through a longitudinal slit that was positioned posterior to the protrusion and flagellar apparatus. Batteries of extrusomes were present within the cytoplasm and had ejection sites through pores in the cell wall. The SSU rDNA phylogeny demonstrated a very close relationship between the benthic P. grandis n. sp. and the planktonic Cryothecomonas longipes. These ultrastructural and molecular phylogenetic data for Protaspis indicated that the current taxonomy of Protaspis and Crythecomonas is in need of re‐evaluation. The composition and identity of Protaspis is reviewed and suggestions for future taxonomic changes are presented. Problems within the genus Cryothecomonas are highlighted as well, and the missing data needed to resolve ambiguities between the two genera are clarified.

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.

Morphology and taxonomy of six marine sand-dwelling Amphidiniopsis species (Dinophyceae, Peridiniales), four of them new, from the German Bight, North Sea

Abstract Six marine species, four of them new, of the dinoflagellate Amphidiniopsis are described from intertidal sand. Amphidiniopsis hirsutum (Balech) Dodge has the plate formula Po 4′ 2a 7″ 8c 4s? 5′″ 2″″. The first scanning electron microscopy observations of Amphidiniopsis swedmarkii (Balech) Dodge are reported and the plate formula Po 4′ la 7″ 6c? 5s? 5′″ 2″″ is established. Amphidiniopsis arenaria Hoppenrath sp. nov. is flattened laterally with a cell size of 30.0–43.5 μm (37.8 ± 4.3 μm, mean ± s) long and 27.0–38.2 μm (33.8 ± 4.5) wide (dorsoventrally). The plate formula is Po 4′ 3a 7″ 6c? 4s? 5′″ 2″″. Amphidiniopsis dentata Hoppenrath sp. nov. is flattened laterally, 38.8–46.5 μm (43.0 ± 2.6) long and 33.7–38.8 μm (37.3 ± 1.7) wide (dorsoventrally). The plate formula is Po 4′ 3a 8″ 6c? 3s? 5′″ 2″″. Amphidiniopsis galericulata Hoppenrath sp. nov. is flattened laterally, 20.3–29.0 μm (24.9 ± 2.5) long and 18.0–25.4 μm (21.9 ± 2.7) wide (dorsoventrally). The plate formula is Po 4′ 3a 7(6)″ ?c 3s? 5′″ (lp) 2″″. Amphidiniopsis cristata Hoppenrath sp. nov. is flattened dorsoventrally, 23.1–34.0 μm (28.9 ± 3.9) long and 19.4–28.6 μm (23.6 ± 2.7) wide (laterally). The plate formula is Po 4′ la 6″ 4c? 4s? 6′″ 2″″. All species have a slightly ascending cingulum.

Morphology and taxonomy of the marine sand-dwelling genus Thecadinium (Dinophyceae), with the description of two new species from the North German Wadden Sea

Abstract Five marine sand-dwelling species of the dinoflagellate genus Thecadinium have been examined from intertidal and subtidal sand and illustrated with light and scanning electron microscopy. Additional information on the known species is provided. The type species, Thecadinium kofoidii, has the plate formula P 3′ 1a 4″ 5(6?)c 5s 4′″ 1″″ the sulcal plates and one more postcingular plate are described. Thecadinium kofoidii is so far the only Thecadinium species possessing chloroplasts. The first light microscopical observations of Thecadinium neopetasatum are reported, and the plate formula is emended to P 3′ 1a 6″ 6c ?s 5′″ 1 ″″, with one more epitheca and two more hypotheca plates. The first scanning electron microscopical observations of Thecadinium dragescoi are reported, and the plate formula is emended to P 5′ 2a 8″ 6(7?)c ?s 4′″ x 2p 1″″, with two newly described apical intercalary plates and the ‘x’ plate. Two species are described as new. Thecadinium ornatum Hoppenrath sp. nov. is flattened laterally with a cell size of 45.0–55.5 μm long and 39.5–46.5 μm wide, a length/width ratio of 1.11–1.22 and a descending cingulum. The plate formula is 4′ 6″ 6c 6s 5′″ 1 ″″. Thecadinium acanthium Hoppenrath sp. nov. is flattened laterally with a cell size of 50.0–64.0 μm long and 37.5–51.0 μm wide, with a length/width ratio of 1.20–1.35 and a descending cingulum. The plate formula is 4′ 6″ 6c ?s 5′″ 1″″.

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

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.

PHYLOGENETICS OF RHINODINIUM BROOMEENSE GEN. ET SP. NOV., A PERIDINIOID, SAND-DWELLING DINOFLAGELLATE (DINOPHYCEAE)1

The phylogeny of Rhinodinium broomeense, a new genus and species of heterotrophic peridinioid dinoflagellates, has been studied based on morphological and molecular genetic data. The genus was found in tidal marine sand habitats in Broome, north‐western Australia, and from three marine sand habitats in Japan. The thecal plate formula is Po 3′ 1a 5″ 4c ?s 5″′ 1″″. A large apical hook points toward the dorsal side. Its plate pattern is similar to species of the genus Roscoffia; however, it differs from that genus in its much larger epitheca, narrow cingulum, which could be interpreted as incomplete, the narrow sulcus without sulcal lists on both sides, and the strong oblique lateral compression. Phylogenetic analyses using partial LSU rDNA sequences, as well as plate pattern information, support the placement of this genus in the Peridiniales; however, it is sufficiently different from other genera that the family affinity remains unclear.

Life Cycle of the pseudocolonial dinoflagellate Polykrikos kofoidii (Gymnodiniales, Dinoflagellata).

The athecate, pseudocolonial polykrikoid dinoflag‐ellates show a greater morphological complexity than many other dinoflagellate cells and contain not only elaborate extrusomes but sulci, cinguli, flagellar pairs, and nuclei in multiple copies. Among polykrikoids, Polykrikos kofoidii is a common species that plays an important role as a grazer of toxic planktonic algae but whose life cycle is poorly known. In this study, the main life cycle stages of P. kofoidii were examined and documented for the first time. The formation of gametes, 2‐zooid‐1‐nucleus stages very different from vegetative cells, was observed and the process of gamete fusion, isogamy, was recorded. Karyogamy followed shortly after completed plasmogamy. A complex reorganization of furrows (cinguli and sulci) and flagella followed zygote formation, resulting in a 4‐zooid zygote with one nucleus. The fate of zygotes under different nutritional conditions was also investigated; well‐fed zygotes were able to reenter the vegetative cycle via meiotic divisions as indicated by nuclear cyclosis. However, nuclear cyclosis was preceded by a presumably mitotic division of the primary zygote nucleus which by definition would imply that P. kofoidii has a diplohaplontic life cycle. Nuclear cyclosis in germlings hatched from spiny resting cysts indicate that these cysts are of zygote origin (hypnozygotes). Hypnozygote formation, cyst hatching, the morphology of the germling (a 1‐zooid cell), and its development into a normal pseudocolony are documented here for the first time. There is evidence that P. kofoidii has a system of complex heterothallism.