J. Eukaryot. Microbiol., 53(5), 2006 pp. 327–342 r 2006 The Author(s) Journal compilation r 2006 by the International Society of Protistologists DOI: 10.1111/j.1550-7408.2006.00110.x Dinoflagellate, Euglenid, or Cercomonad? The Ultrastructure and Molecular Phylogenetic Position of Protaspis grandis n. sp. MONA HOPPENRATH and BRIAN S. LEANDER Canadian Institute for Advanced Research, Program in Evolutionary Biology, Departments of Botany and Zoology, University of British Columbia, Vancouver, BC, Canada V6T 1Z4 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 mm long and 20.0–35.0 mm 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. Key Words. Cercozoa, Cryothecomonas, morphology, phylogenetic analysis, Protaspis, SSU rDNA, taxonomy, ultrastructure. HE genus Protaspis was described by Skuja (1939), with the Protaspis species occur in marine benthic habitats, freshwater, T type species Protaspis glans and two additional species, and marine plankton communities, and soil (Auer and Arndt 2001; Protaspis maior and Protaspis metarhiza (Skuja 1939). Skuja Ekelund and Patterson 1997; Ekebom, Patterson, and Vrs 1995/ (1939) erected the new family Protaspidaceae for his new genus 96; Larsen 1985; Larsen and Patterson 1990; Lee and Patterson and classified it tentatively within the euglenids, because of the 2000; Lee et al. 2003, 2005; Norris 1961; Patterson et al. 1993; strong continuous ‘‘periplast,’’ the ventral longitudinal furrow, Skuja 1939, 1948; Tong et al. 1998; Vrs 1992, 1993). Currently, the two heterodynamic flagella, the large nucleus with nucleolus the genus contains 10 species: P. glans Skuja 1939, P. maior and the paramylon-like reserve product. The flagella insert in the Skuja 1939, P. metarhiza Skuja 1939, P. obovata Skuja 1948, anterior part of the ventral furrow and are clearly separated from Protaspis tanyopsis Norris 1961, Protaspis gemmifera Larsen and each other. The movement is by gliding and food uptake is by Patterson 1990, Protaspis obliqua Larsen and Patterson 1990, pseudopods formed out of the furrow (Skuja 1939). Skuja (1948) Protaspis tegere Larsen and Patterson 1990, Protaspis verrucosa described a fourth species, Protaspis obovata. The large nucleus Larsen and Patterson 1990, and Protaspis simplex Vrs 1992. The of this species had the characteristic morphology of a dinoflagel- distinguishing features among some of these species are not clear, late nucleus (dinokaryon), which led Skuja (1948) to reclassify and it is likely that some Protaspis species will prove to be Protaspis within the Pyrrophyta (dinoflagellates). This unusual conspecific (for detailed discussion in Lee 2001). The unresolved combination of characters led Skuja (1948) to entertain the phylogenetic position of Protaspis and the potential affiliation to possibility that protaspids might occupy an intermediate phylo- dinoflagellates or euglenids motivated us to investigate the phy- genetic position between euglenids and dinoflagellates. logeny of a new species, Protaspis grandis n. sp., on the basis of From the time of Skuja’s work to the early 1990s, Protaspis ultrastructural and small subunit (SSU) rDNA sequence data. was consistently treated as a dinoflagellate taxon (Chre´tiennot- Dinet et al. 1993; Loeblich 1969; Loeblich and Loeblich 1966; MATERIALS AND METHODS Silva 1980; Sournia 1973, 1978, 1986, 1993). Fensome et al. (1993) excluded Protaspis from the division Dinoflagellata and Collection of organisms. Samples were collected with a stated that it is a problematic genus, possibly of euglenid affinity. spoon during low tide at Centennial Beach, Boundary Bay, BC, However, at about that same time, Protaspis was also tentatively Canada. The salinity of the water is about 30–33 psu. The sand classified as belonging to the Thaumatomastigaceae/Thaumato- samples were transported directly to the laboratory and the mastigidae (Larsen and Patterson 1990; Patterson et al. 2002; flagellates were separated from the sand by extraction through a Patterson and Zo¨lffel 1991). Mylnikov and Karpov (2004) argued 45-mm filter using melting seawater-ice (Uhlig 1964). The flagel- that Protaspis should be transferred into the Cercomonadida lates accumulated in a Petri dish beneath the filter and were then because protaspids do not have a flagellar pocket and unlike identified with an inverted-microscope at 40 Â –250 Â magnifi- thaumatomonads, do not have body scales. This opinion is cation. Cells were isolated by micropipetting and used directly reflected in the latest higher-level classification of eukaryotes, (not from culture) for the preparations described below. where Protaspis is classified in the Cercomonadida in the Light microscopy. Cells were observed directly and micro- ‘‘Family’’ Heteromitidae (Adl et al. 2005). manipulated with a Leica DMIL inverted microscope. For differ- ential interference contrast light microscopy, micropipetted cells were placed on a glass specimen slide and covered with a cover Corresponding Author: M. Hoppenrath, Canadian Institute for Ad- slip. Images were produced with a Zeiss Axioplan 2 imaging vanced Research, Program in Evolutionary Biology, Departments of microscope connected to a Leica DC500 color digital camera. Botany and Zoology, University of British Columbia, Vancouver, BC, Scanning electron microscopy. A mixed-extraction sample Canada V6T 1Z4—Telephone number: 604-822-4892; FAX number: was fixed overnight with two drops of acidic Lugol’s solution. 604-822-6089; e-mail: [email protected] Cells were transferred onto a 5-mm polycarbonate membrane filter 327 328 J. EUKARYOT. MICROBIOL., VOL. 53, NO. 5, SEPTEMBER– OCTOBER 2006 (Corning Separations Div., Acton, MA), washed with distilled We also examined the dataset with Bayesian analysis using the water, dehydrated with a graded series of ethanol and critical point program MrBayes 3.0 (Huelsenbeck and Ronquist 2001). The dried with CO2. Filters were mounted on stubs, sputter-coated program was set to operate with GTR, a g distribution and four with gold and viewed under a Hitachi S4700 Scanning Electron MCMC chains starting from a random tree (default tempera- Microscope (SEM). Some SEM images were presented on a black ture 5 0.2). A total of 2,000,000 generations were calculated with background using Adobe Photoshop 6.0 (Adobe Systems, San trees sampled every 100 generations and with a prior burn-in of Jose, CA). 200,000 generations (2,000 sampled trees were discarded). A Transmission electron microscopy. Cells were concentrated majority rule consensus tree was constructed from 16,000 post- in a microfuge tube by micropipetting and slow centrifugation. burn-in trees with PAUPÃ 4.0. Posterior probabilities correspond The pellet of cells was prefixed with 2% (v/v) glutaraldehyde in to the frequency at which a given node is found in the post-burn-in seawater at 4 1C for 30 min. Cells were washed twice in filtered trees. seawater (30–35 psu) before post-fixation in 1% (w/v) OsO4 in GenBank accession numbers. Allas diplophysa (AF411262), seawater for 30 min at room temperature. Cells were dehydrated Allas sp. (AF411263), Bigelowiella natans (AF054832), Cerco- through a graded series of ethanol, infiltrated with acetone–resin monas longicauda (AF101052), Cercomonas sp. (U42448), Cer- mixtures (pure acetone, 2:1, 1:1, 1:2, pure resin), and embedded in cozoa sp. WHO1 (AF411273), Chlorarachnion reptans (U03477), pure resin (Epon 812). The block was polymerized at 60 1C and Cryothecomonas aestivalis (AF290541), Cryothecomonas long- sectioned with a diamond knife on a Leica Ultracut UltraMicro- ipes (AF290540), Euglypha rotunda (AJ418784), Gromia ovifor- tome. Thin sections were post-stained with uranyl acetate and lead mis (AJ457813), Heteromita globosa (U42447), Lotharella citrate and viewed under a Hitachi H7600 Transmission Electron globosa (AF076169), Massisteria marina (AF174372), Paulinella Microscope. chromatophora (X81811), Phagomyxa bellerochea (AF310903),