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 , 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. 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 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. , 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 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 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), 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), rotunda (AJ418784), 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), marina (AF174372), Microscope. chromatophora (X81811), Phagomyxa bellerochea (AF310903), DNA extraction, amplification, cloning and squencing. Forty Polymyxa betae (AF310902), P. grandis n. sp. (DQ303924), individually isolated (uncultured) cells were washed three times in Pseudodifflugia cf. gracilis (AJ418794), Pseudopirsonia mucosa filtered (-free) seawater. Genomic DNA was extracted (AJ561116), Thaumatomastix sp. (AF411261), from the 40-cell preparation using a standard hexadecyltrimethy- seravini (AF411259), Thaumatomonas sp. (U42446), Thaumato- lammonium bromide (CTAB) extraction protocol (Zolan and monas sp. (SA) (AF411260), uncultured cercozoan (AF372764), Pukkila 1986). The PCR amplification protocol using universal uncultured cercozoan (AY180012), uncultured cercozoan eukaryotic primers reported previously (Leander, Clopton, and (AY180035), uncultured cercozoan (AY620274), uncultured cer- Keeling 2003) consisted of an initial denaturing period (95 1C cozoan (AY620276), uncultured cercozoan (AY620277), uncul- for 2 min); 35 cycles of denaturing (92 1C for 45 s), annealing tured cercozoan (AY620279–AY620281), uncultured cercozoan (48 1C for 45 s), and extension (72 1C for 1.5 min); and a final (AY620294), uncultured cercozoan (AY620295), uncultured cer- extension period (72 1C for 5 min). Polymerase chain reaction cozoan (AY620300), uncultured cercozoan (AY620314), uncul- products corresponding to the expected size were gel isolated tured cercozoan (AY620316), uncultured cercozoan (AY620320– and cloned into the pCR2.1 using the TOPO TA cloning kit AY620323), uncultured cercozoan (AY620340), uncultured (Invitrogen, Burlington, ON, Canada). Two clones were sequenced cercozoan (AY620348–AY620353), uncultured cercozoan with ABI big-dye reaction mix using the vector primers and (AY620355), uncultured cercozoan (AY620357), uncultured internal primers oriented in both directions. eukaryote TAGIRI-2 (AB191410), uncultured eukaryote Phylogenetic analyses. The Protaspis SSU rDNA sequence (AJ130856), uncultured eukaryote (AY082998). was aligned with other eukaryotic sequences using MacClade 4 (Maddison and Maddison 2000) forming a 54-taxon alignment. Maximum likelihood (ML), ML distance, and Bayesian methods under different DNA substitution models were performed. All RESULTS gaps were excluded from the alignments before phylogenetic Cell shape and flagella. The flagellated stage in the life cycle analysis. The a shape parameters were estimated from the data of P. grandis was investigated. Plasmodial and cyst stages were using HKY under a g distribution plus invariable sites model and not observed. Cells were 32.5–55.0 mm long, 20.0–35.0 mm wide four rate categories (a 5 0.28, Ti/Tv 5 1.38, fraction of invariable (n 5 21), dorso-ventrally flattened and shaped like elongated ovals sites 5 0.08). A gamma-corrected ML tree (analyzed using the with parallel lateral sides (Fig. 1–4). The ventral side of the cell parameters listed above) was constructed with PAUPÃ 4.0 using was concave and the dorsal side was slightly convex (Fig. 5). Two the general time reversible (GTR) model for base substitutions thin, heterodynamic flagella emerged from separate subapical (Posada and Crandall 1998; Swofford 1999). Gamma corrected depressions on the ventral side of the cell (Fig. 4, 11). The anterior ML tree topologies found with HKY and GTR were identical. flagellum was about half a cell length, and the trailing flagellum Maximum likelihood bootstrap analyses were performed in was about one cell length. A protrusion emerging from the right PAUPÃ 4.0 (Swofford 1999) on 100 re-sampled datasets under furrow border pointed to the left and separated the insertion an HKY model using the a shape parameter, proportion of depressions of the flagella (Fig. 1, 4, 11, 15). The posterior invariable sites and transition/transversion ratio (Ti/Tv) estimated flagellum inserted below the protrusion and was positioned from the original dataset. slightly to the left. A narrow ventral furrow is positioned medially Maximum likelihood distances for the SSU rDNA dataset and extends about one-third of the cell length posteriorly from the were calculated with TREE-PUZZLE 5.0 using the GTR sub- insertion point of the posterior flagellum and towards the posterior stitution matrix (Strimmer and Von Haeseler 1996). A distance end (Fig. 1, 3, 4, 11). The flagella were anchored within flagellar tree was constructed with weighted neighbor joining (WNJ) pits and emerged through funnels with a sharp outer margin and using Weighbor (Bruno, Socci, and Halpern 2000). Five-hundred ring-shaped basal depression (Fig. 16). Transmission electron bootstrap datasets were generated with SEQBOOT (Felsenstein micrographs through this region of the cell contained a structure 1993). Respective distances were calculated with the shell that lacked the typical 912 organization of microtubules and was script ‘‘puzzleboot’’ (M. Holder and A. Roger, www.tree-puz- composed of material arranged in concentric rings of different zle.de) using the a shape parameter and proportion of invariable density (Fig. 17). This morphology is consistent with both a sites estimated from the original dataset and analyzed with flagellum, in transverse view, as seen in C. aestivalis (Drebes Weighbor. et al. 1996, Fig. 23) and an extrusome, in transverse view. HOPPENRATH & LEANDER—MORPHOLOGY AND PHYLOGENY OF PROTASPIS GRANDIS 329

Fig. 1–10. Light micrographs of Protaspis grandis n. sp. (1–5) and Protaspis obliqua (6–9). 1. Ventral view of P. grandis n. sp. showing elongated oval cell shape, granular cytoplasm, ventral furrow near the anterior end (arrowhead), and an anterior protrusion (arrow) stemming from the right margin of the furrow. 2. Ventral view, median focus, of P. grandis n. sp. showing a thickened cell wall (arrow), lipid globules near the posterior end of the cell (double arrowhead), and a circular, granular nucleus positioned in the posterior half of the cell (n). 3. Ventral view of P. grandis n. sp. showing a closed ventral furrow (arrowhead). 4. Ventral view of P. grandis n. sp. showing the nucleus (n), an open ventral furrow, and the insertion point of the posterior flagellum (arrowhead) within a depression that is positioned below the anterior protrusion (arrow). 5. Lateral view of P. grandis n. sp. showing the concave ventral surface, convex dorsal surface, granular nucleus with permanently condensed chromosomes (n), and the thickened cell wall (arrow). 6. Ventral view, median focus, of P. obliqua showing the posterior indentation (arrowhead) and the thickened cell wall (arrow). 7. Ventral view, median focus, of P. obliqua showing the nucleus positioned in the anterior half of the cell (n) and the thickened cell wall (arrow). 8. Ventral view of P. obliqua showing the posterior indentation (arrowhead). 9. Ventral view of P. obliqua showing the anterior indentation (arrow). 10. An unidentified Protaspis species showing the typical morphology and distribution of pseudopods over the substrate. (Fig. 1–5, Bar 5 10 mm; Fig. 6–9, Bar 5 10 mm; Fig. 10, Bar 5 10 mm).

Nucleus and cytoplasm. A large, round nucleus with a gran- division, the flagellar apparatus doubled (Fig. 26). Although cell ular appearance was situated in the posterior half of the cell and division was not observed during this study, the pattern of flagellar midway between the lateral margins (Fig. 2, 4, 5). The nucleus replication indicated that longitudinal division most likely occurs was never observed in a lateral position or in the anterior half of along a longitudinal cleavage furrow, which has also been de- the cell. Nuclear caps were not observed. The nucleus contained a scribed for other species in the genus. large nucleolus and permanently condensed chromosomes dis- Cell wall. The cell surface was smooth and reinforced with a tributed evenly (Fig. 12, 13). Some of our micrographs showed a thickened cell wall comprised of at least seven different layers of convoluted cleft in the anterior side of the nucleus (Fig. 13). The material having different thicknesses, densities, and architectures cytoplasm was generally colorless and contained food , (Fig. 2, 3, 11, 12, 15, 27, 28). The multilayered wall was secreted masses of small vesicles/granules, and often some larger colored outside of the plasma membrane (Fig. 28). An amorphous ‘‘basal particles (e.g. yellow, orange, red, or brown) (Fig. 1–4, 12, 13). layer’’ about 150 nm thick was positioned directly above the The ultrastructure of the large colored particles was uniform and plasma membrane and was overlain by a broad ‘‘intermediate often darkly stained suggesting that they were globules of lipids layer’’ about 400 nm thick (Fig. 28). A ‘‘vesicular layer’’ (50 nm (Fig. 12, 13). Mitochondria were evenly distributed throughout thick) interrupted the relatively homogeneous intermediate layer the cytoplasm and had tubular-shaped cristae (Fig. 14). The near its superficial margin (Fig. 28). A thin electron-dense ‘‘outer cytoplasm also contained elongated Golgi bodies composed of lamina’’ was squeezed between the intermediate layer and a stacks of about six to nine cisternae (Fig. 20). sealing ‘‘coat’’ consisting of three ridged layers: a darker ‘‘deep Ventral furrow and pseudopodia. In some cells, relatively coat,’’ a columnar ‘‘mid-coat,’’ and a parallel ‘‘superficial coat’’ long and thin pseudopodia emerged from the ventral furrow and (Fig. 27, 28). In tangential section, the regular spacing of the spread over the substrate surface (not shown). An unidentified ridges of the coat were easily discernable (Fig. 29). Cylindrical Protaspis cell with typical extruded pseudopodia is shown in Fig. pores pierced the basal and intermediate layers and permitted 10. Pseudopodia were evident in our P. grandis samples prepared extrusomes to be discharged through the cell wall (Fig. 27, 30– for SEM and TEM as spherical globules located near the ventral 32). Pores were never observed piercing the tri-layered coat (Fig. furrow (Fig. 18, 19). The ventral furrow was observed in three 27). Batteries of extrusomes oriented in the same direction were different states: closed (Fig. 21), open (Fig. 22), and protruding usually concentrated in clusters (Fig. 31), and each extrusome was (Fig. 23). A distinctive slit was located inside the ventral furrow, located within a membrane-bound vesicle (Fig. 32). from which the pseudopodia emerged (Fig. 19, 22–24). The Molecular phylogeny. We sequenced the SSU rRNA gene pseudopodia were bordered by the plasma membrane, lacked a from a multi-specimen sample of P. grandis collected in Septem- cell wall, and were rich in mitochondria (Fig. 25). Before cell ber 2004. Basic local alignment search tool results robustly placed 330 J. EUKARYOT. MICROBIOL., VOL. 53, NO. 5, SEPTEMBER– OCTOBER 2006 HOPPENRATH & LEANDER—MORPHOLOGY AND PHYLOGENY OF PROTASPIS GRANDIS 331 the sequence together with C. longipes and several related envir- the cell an asymmetrical appearance (Fig. 34). Moreover, the onmental sequences, all of which belonged to the ‘‘Cercozoa.’’ furrow of P. obliqua is in the posterior half of the cell and We created a 54-taxon alignment (1,006 unambiguous sites) the nucleus is positioned in the anterior half, which stands in consisting of ingroup cercozoans and representative sequences contrast to the anterior furrow and posterior nucleus position from plasmodiophorids, , and Gromia (Fig. in P. grandis (Larsen and Patterson 1990, 2000; Table 1; 33). Protaspis grandis, C. longipes, and three environmental Lee 2001 and Fig. 34). These morphological characters clearly sequences (AY620323, AY620340, and AY620352) branched distinguish P. obliqua from P. grandis, and we were always able together with strong support from both bootstrap statistics and to distinguish these two species in samples in which both of these Bayesian posterior probabilities (Fig. 33). This subclade fell species co-occurred (Fig. 1–9). The P. obliqua cells were 27.5– within a much larger clade containing C. aestivalis and several 35.0 mm long and 25.0–32.5 mm wide (n 5 10), which is slightly environmental sequences, all of which formed the well-supported larger than that reported in the literature (Table 1). No intermedi- clade. Interestingly, C. longipes branched much ate morphologies between P. obliqua and P. grandis were ob- more closely to P. grandis than to C. aestivalis (Fig. 33). None- served in our samples. Therefore, we establish Protaspis grandis theless, the Cryomonadida clade formed the sister group to n. sp. and provide the following species description. a sequence clade of unknown identity, namely ‘‘undescribed cecozoan clade II’’ and was only distantly related to the Thau- Taxon Description matomonadida (Fig. 33). Cercozoa Cavalier-Smith 1998, emend. Adl et al. 2005 Cercomonadida Poche 1913, emend. Vickerman 1983, emend. DISCUSSION Mylnikov 1986 Heteromitidae Kent 1880, emend. Mylnikov 1990, emend. Comparative morphology. The overall light-microscopical Mylnikov and Karpov 2004 appearance of the species described here conforms with the Protaspis Skuja 1939 characteristic features for the genus Protaspis: heterodynamic Protaspis grandis Hoppenrath et Leander n. sp. flagella insert subapically within separate depressions on the ventral surface of the cell, movement is by substrate-mediated Diagnosis. Cells shaped as elongated ovals with parallel lateral gliding, feeding is by means of pseudopodia that emerge from a sides, dorsoventrally flattened, 32.5–55.0 mm long, 20.0–35.0 mm ventral furrow and the granularity of the nucleus is clearly visible. wide. Two heterodynamic flagella, inserted subapically, separated Protaspis grandis n. sp. is larger than all other described species in by a protrusion. Flagellar pits with funnel. Ventral longitudinal the genus, but its smallest size overlaps with the largest size range furrow in the anterior half of cell. Nucleus in the posterior half of of the larger species, P. maior, P. metarhiza, and P. obovata cell, with nucleolus and permanently condensed chromosomes. (Table 1). Protaspis grandis n. sp. is clearly distinguishable from Pseudopodia emerge through a slit within the ventral furrow. these species and nearly all other Protaspis species by having an Thickened cell wall outside of the plasma membrane consisting anterior protrusion that separates the flagellar insertion points and of seven layers: basal layer, intermediate layer, vesicular layer, an obviously thick cell wall (Fig. 34). Only P. tanyopsis and P. outer lamina, deep coat, mid-coat, and superficial coat. Basal layer obliqua have a similar protrusion (Larsen and Patterson 1990; Lee and intermediate layer pierced by pores for the discharge of and Patterson 2000; Lee, Simpson, and Patterson 2005; Norris extrusomes. 1961). Protaspis tanyopsis also has a similar shape to P. grandis Holotype/type micrograph. Fig. 4. but is smaller, only slightly flattened dorso-ventrally, and lacks a Type locality. Tidal sand-flat at Centennial Beach, Boundary thickened cell wall. Moreover, P. tanyopsis is longer than P. Bay, BC, Canada. grandis relative to the cell width and the nucleus is positioned The species was observed with higher abundance in September anteriorly rather than posteriorly (Norris 1961; Table 1 and Fig. 2004 and 2005, but also in October 2004, March, April, June, and 34). Norris (1961) described P. tanyopsis as having a small lobe August 2005 in low cell numbers. near the anterior end of the groove margin but did not specify Habitat. Marine. which margin, the left or right side of the groove. Although we Etymology for the specific epithet. Refers to the large cell remain doubtful, his drawing suggests that the lobe/protrusion is size relative to all other known species within the genus. part of the left-hand groove margin (Fig. 34). By contrast, the anterior protrusion of P. grandis and P. obliqua emerges from the Which characters are useful for the recognition of different right-hand furrow margin. Like P. grandis, P. obliqua also has a species? Vrs (1992) stated that the species are ‘‘. . . distinguished thickened cell wall (Larsen and Patterson 1990; Lee 2001; Lee by size and shape, the length and path of the groove, and the and Patterson 2000; Lee et al. 2005). However, the cells of P. presence or absence of (1) a nuclear cap, (2) a protrusion separat- obliqua are significantly smaller than P. grandis, are round to oval ing the flagella, (3) rod-shaped bodies of reserve material [and] . . . rather than elongated, and have a posterior indentation that gives Protaspis verrucosa . . . is distinguished by the globular cell body b—————————————————————————————————————————————————————— Fig. 11–17. Scanning (SEM) and transmission electron (TEM) micrographs of Protaspis grandis n. sp. 11. SEMs showing a dorsal surface view (left- hand cell) and a ventral surface view (right-hand cell) containing the anterior protrusion (double arrowhead) and longitudinal ventral slit (arrow); the ventral furrow is not present due to the swollen state of the cell (Bar 5 5 mm). The absence of flagella is a common artifact in SEM preparation. 12. Sagittal TEM showing the thickened cell wall (arrow), posterior nucleus with permanently condensed chromosomes and conspicuous nucleolus, large lipid globules (lg) posterior to the nucleus, and vesicular cytoplasm (v) anterior to the nucleus (Bar 5 2 mm). 13. TEM showing a nucleus with a convoluted cleft (arrow), a food (fv), and darkly stained lipid globules (l) (Bar 5 2 mm). 14. TEM showing a typical double membrane-bound with tubular cristae (Bar 5 0.25 mm). 15. SEM showing the anterior protrusion (double arrowhead) separating the depressions within which the heterodynamic flagella originate (arrows). The shortness of flagella is a common artifact in SEM preparation. The ventral slit (arrowhead) is evident within the ventral furrow (Bar 5 2 mm). 16. SEM showing a funnel (arrowhead) from which a short flagellum (arrow) emerges (Bar 5 0.5 mm). 17. TEM showing a membrane-bound structure (arrowheads) near the ventral slit composed of material having differing densities and arranged in concentric rings. This morphology is consistent with both a flagellum, in transverse view, as seen in Cryothecomonas aestivalis and an extrusome, in transverse view (Bar 5 0.1 mm). 332 J. EUKARYOT. MICROBIOL., VOL. 53, NO. 5, SEPTEMBER– OCTOBER 2006 HOPPENRATH & LEANDER—MORPHOLOGY AND PHYLOGENY OF PROTASPIS GRANDIS 333 and a warty cell surface’’ (p. 91). It has been noticed that the size Pseudopodia are also difficult to observe and the morphological range of Protaspis species was often expanded when further details associated with these structures are not available for all the observations of a species were made in different habitats (e.g. species, which questions the taxonomic value of these features. Lee 2001; Lee and Patterson 2000). Most of the Protaspis species Therefore, in our view, characters like anterior protrusions, ante- show considerable overlap in their size ranges (Table 1), and rior or posterior indentations, furrow morphology, and the relative therefore cell size is only of value when the differences are thickness of the cell wall are the best diagnostic features currently relatively discrete and are used in connection with other features. available. In addition, the position of the nucleus within the cell The flattened cell shape of most of Protaspis species is very (e.g. anterior versus posterior) and cell shape (round versus similar (round to oval to ovoid; Table 1 and Fig. 34), and most elongated) used in connection with discrete differences in the likely there is a limited degree of variation (Table 1) depending range of cell sizes appear to be practical ‘‘combination- on the age of the cell, feeding stages and cell cycle stages characters’’ (Fig. 34). (e.g. P. obovata in Skuja 1948). The cell shape must differ After taking the above discussion about the usefulness of significantly in order to be a distinguishing character by itself certain characters into account, there is the strong possibility that (e.g. round in contrast to elongated oval). P. glans, P. maior, P. metarhiza, and P. tegere are actually The morphology of the ventral furrow seems to be a useful conspecific. Lee (2001) also discussed this possibility and also character, even if the path and visibility varies in older cells (e.g. included P. obovata to the list. We think that the apex morphology P. maior in Skuja 1939). The length of the furrow in relation to the with a papilla-like structure in combination with the truncated ant- cell length, the width (e.g. broad versus widening at the ends apex characterizes P. obovata, and we therefore do not regard it as versus narrow), and the position (e.g. indistinct versus whole cell synonymous. Protaspis gemmifera and P. simplex are probably length versus mainly anterior versus mainly posterior) of the also synonymous. Perhaps, the conspecificity of these taxa should furrow appear to be reasonable diagnostic characters (Table 1). be demonstrated with molecular methods before taxonomic The presence or absence of nuclear caps is not a consistent feature changes are proposed. Nonetheless, it is probable that the genus or has not been recorded reliably (Tong et al. 1998); P. gemmifera, will ultimately be reduced from eleven to seven species: P. glans, P. tegere, and P. verrucosa were all described both with and P. obovata, P. tanyopsis, P. gemmifera, P. verrucosa, P. obliqua, without them (Larsen and Patterson 1990; Lee 2001; Lee and and P. grandis. It appears that other species of Protaspis have Patterson 2000; Patterson et al. 1993; Tong et al. 1998). The been observed but not formally described. For instance, Sournia anterior protrusion separating the flagella is still regarded as a (1986) reported a light micrograph of two cells that was referred consistent species character. Special cell inclusions like conspic- to as Protaspis of unknown identity (p. 143, Fig. 51). Although the uous reserve material (e.g. rod-shaped in P. gemmifera) are no organization of flagellar insertions and pseudopodia were not longer used as a characteristic feature (Lee 2001; Lee and visible, the overall morphology of this cell strongly suggests that Patterson 2000). The presence or absence of warts on the cell it is a Protaspis species with a thick cortex, anterior nucleus and surface is not a constant character or not reliably observed (e.g. P. long ventral furrow. gemmifera, P. tegere and P. simplex; Ekelund and Patterson 1997; Our phylogenetic analyses of P. grandis demonstrates a close Ekebom et al. 1995/96; Larsen and Patterson 1990; Lee 2001; Lee relationship between this species and C. longipes Schnepf et Ku¨hn and Patterson 2000; Lee et al. 2005; Vrs 1992). The position of 2000 (Fig. 33). This result is consistent with ultrastructural the nucleus is nearly the same in all Protaspis species (Table 1 and similarities between the two species. Like P. grandis, C. longipes Fig. 34) and varies to a certain extent in P. metarhiza and P. glans has a multilayered cell wall, a nucleus with a conspicuous depending on the age of the cells (Larsen 1985; Larsen and nucleolus and condensed chromosomes, extrusomes of the same Patterson 1990; Skuja 1939). Protaspis grandis is the only species morphology, pseudopodia that protrude through a preformed slit described so far with the nucleus positioned in the posterior half of and flagella that sit within separated flagellar pits and emerge the cell. The thickness of the cell wall is a distinguishing feature, through distinctive funnels (Schnepf and Ku¨hn 2000; Fig. 12–32). with only P. obliqua and P. grandis having a thick one that is A multilayered cell wall, a nucleus with distinct areas of easily visible with light microscopy (Larsen and Patterson 1990; condensed chromatin and flagella emerging through funnels are Lee and Patterson 2000; Fig. 1–9). ultrastructural characters that have been used to diagnose the A posterior (and anterior?) indentation seems to be a special genus Cryothecomonas (Thomsen et al. 1991; Table 1). feature, so far only described for P. obliqua (Larsen and Patterson Nonetheless, there are clear ultrastructural differences between 1990; Lee 2001; Lee and Patterson 2000). Moreover, there is large P. grandis and C. longipes, such as the organization of the and overlapping range for flagellar lengths within a species (Table flagellar insertion points, the abundance and distribution of con- 1), making it useless for species identification. The mode of densed chromosomes, the presence of a ventral furrow and the flagellar movement could be of interest, but this behavioral relative thicknesses of cell wall layers. The anterior flagellum is character has not been sufficiently documented for all of the inserted apically and the posterior flagellum inserted subapically described species. The possibility of different cell behaviors in C. longipes (Schnepf and Ku¨hn 2000), whereas both flagella are during different feeding and cell cycle stages would make the inserted subapically in all Protaspis species (Table 1 and Fig. 34). use of these characteristics for species separation challenging. These different conformations of the flagellar apparatus likely

b—————————————————————————————————————————————————————— Fig. 18–26. Scanning (SEM) and transmission electron (TEM) micrographs of Protaspis grandis n. sp. 18. SEM showing a ventral surface view containing the anterior protrusion (double arrowhead) and spherical globules of pseudopodia (arrowhead) emerging from the ventral furrow (arrow) (Bar 5 5 mm). 19. Sagittal TEM showing the anterior protrusion (double arrowhead), the ventral slit (arrow), and spherical globules of pseudopodia (arrowheads) (Bar 5 4 mm). 20. TEM showing an elongated Golgi body (dictyosome) containing seven stacked cisternae (Bar 5 0.25 mm). 21. SEM showing the ventral furrow in the closed state (Bar 5 0.5 mm). 22. SEM showing the ventral furrow in the open state containing the ventral slit (arrowheads) (Bar 5 0.5 mm). 23. SEM showing the swollen ventral slit (arrowhead) (Bar 5 1 mm). 24. TEM showing a tangential view of the ventral slit (Bar 5 0.5 mm). 25. TEM of a pseudopodium showing a large population of mitochondria with tubular cristae (m) (Bar 5 1 mm). 26. SEM of a cell preparing for division showing the anterior protrusion (double arrowhead) separating the replicated anterior flagella (arrows) and the replicated insertion funnels (arrowheads) for the posterior flagella (Bar 5 1 mm). 334 J. EUKARYOT. MICROBIOL., VOL. 53, NO. 5, SEPTEMBER– OCTOBER 2006 HOPPENRATH & LEANDER—MORPHOLOGY AND PHYLOGENY OF PROTASPIS GRANDIS 335 reflect different swimming modes associated with the benthic and and Cryothecomonas vesiculata, share these features (Thomsen planktonic lifestyles of most Protaspis and Cryothecomonas et al. 1991; Table 1). Cryothecomonas aestivalis Drebes, Ku¨hn, species, respectively. The nucleus of C. longipes is lobed, with and Schnepf, 1996 has apically inserting heterodynamic flagella a prominent nucleolus surrounded by heterochromatin aggregated separated by a papilla, and the pseudopodia emerge from the near the periphery of the nucleus (Schnepf and Ku¨hn 2000), posterior cell pole (Drebes et al. 1996). Cryothecomonas longipes whereas the heterochromatin in P. grandis is more abundant and has the anterior flagellum inserting apically, the posterior flagel- evenly distributed in a non-lobed nucleus. Unlike P. grandis,the lum inserting subapically (heterodynamic), and a rarely noticeable ventral slit in C. longipes is not situated in a ventral furrow papilla (Schnepf and Ku¨hn 2000; Table 1 and Fig. 34). The (Schnepf and Ku¨hn 2000). Key differences in the multilayered pseudopodia emerge from a ventral slit in the anterior cell half cell wall of P. grandis, when compared with C. longipes are as (Schnepf and Ku¨hn 2000; Table 1). follows: (1) the coat is more stratified containing not only a If we stress the importance of differences in the flagellar columnar mid-coat layer (like C. longipes) but also a deep coat insertion organization and patterns of motility and view them as layer and a parallel superficial coat layer; (2) the intermediate diagnostic characters at the generic level, then the genus Cryothe- layer is significantly thicker and contains a vesicular layer em- comonas should be split into three genera, with C. aestivalis and bedded within it (putatively homologous to the ‘‘compact layer’’ C. longipes each representing a new genus. However, we hesitate of C. longipes); and (3) the basal layer is much thicker (Fig. 12, to formally split the genus at this early stage, because there are no 27–30). Although our naming scheme is consistent with the sequence data yet available for any Cryothecomonas species that descriptions presented for C. longipes (Schnepf and Ku¨hn shares the same characters as the type. The SSU rDNA sequence 2000), we were unable to rule out the possibility that the ‘‘outer for the type species, C. armigera, should be added to the existing lamina’’ in P. grandis actually represents the plasma membrane dataset before nomenclatural changes are recommended. None- (Fig. 27–30). theless, if the current members of Cryothecomonas were split into These relatively modest differences in the basic characters three genera, then they would all be distinguishable from Protas- between these two species raise the question of whether P. grandis pis on the basis of flagellar insertion organization, with the new and C. longipes should be classified within two different genera. ‘‘C. longipes-genus’’ being most closely related to it. Alterna- Moreover, is it sensible for us to generalize these observations to tively, it might be more appropriate to de-emphasize slight all members of both genera? If so, is it warranted to transfer P. modifications of the configuration of flagellar insertions (e.g. grandis and related Protaspis species into the genus Cryotheco- C. longipes versus P. grandis) and transfer all species of Cryothe- monas or, conversely, to transfer C. longipes into the genus comonas into Protaspis, making the former a junior synonym of Protaspis (assuming Cryothecomonas is a junior synonym of the latter. This would be the best approach to take if at some point Protaspis)? Currently, we do not think that we can generalize it is demonstrated that all of these species are intermingled within our ultrastructural results for P. grandis to be characteristic for all a robustly supported clade inferred from SSU rDNA sequences or Protaspis species because there are no further TEM observations a comparable molecular marker. Although there are no data for for the genus. It is highly likely that all species have a nucleus the type species of Protaspis yet, it is likely that all of these with permanently condensed chromosomes, because the granular species whether placed in one or up to four different genera will appearance of the nucleus is readily visible with light microscopy share the following features: (1) mitochondria with tubular cris- (which gave rise to previous classifications of Protaspis within the tae; (2) nucleus with permanently condensed chromosomes and dinoflagellates and euglenids, see the Introduction). The posses- conspicuous nucleolus; (3) multilayered cell wall outside of the sion of a multilayered wall and flagellar insertion funnels should plasma membrane; (4) feeding by means of pseudopodia; and be verified by ultrastructural investigations of the other Protaspis (5) flagella emerging through funnels. species. As circumscribed today, Protaspis seems to be a well- Molecular phylogeny and further expansion of the Cerco- defined genus. zoa. Our phylogenetic analyses demonstrated that P. grandis is However, this is not the case for Cryothecomonas, and we closely related to C. longipes. The sequence from P. grandis argue that it is an artificial (polyphyletic) genus for the following represents only the third reference taxon (morphologically reasons. The type of flagellar insertion, the flagella motion and the described species) within the Cryomonadida clade, which is site of pseudopodia formation are different within the genus currently comprised mainly of unidentified environmental se- (Drebes et al. 1996; Schnepf and Ku¨hn 2000; Thomsen et al. quences (Fig. 33). A phylogenetic analysis excluding the shorter 1991). The type species Cryothecomonas armigera Thomsen et al. environmental sequences and including only morphologically 1991 has apically inserting isodynamic flagella separated by a described taxa within the Cercozoa (including P. grandis)is papilla (Thomsen et al. 1991; Table 1 and Fig. 34). The pseudo- presented elsewhere (Hoppenrath and Leander 2006). Previous podia emerge from a slit () located posterio-laterally SSU rRNA phylogenies have shown that Cryothecomonas clus- (Thomsen et al. 1991; Table 1 and Fig. 34). All other species in ters robustly within the Cercozoa and have helped clarify the the genus that were described together with the type species, relative relationships between Cryothecomonas, Heteromita, namely Cryothecomonas inermis, Cryothecomonas scybalophora, Thaumatomonas, and Cercomonas (Cavalier-Smith and Chao b——————————————————————————————————————————————————————— Fig. 27–32. Transmission electron micrographs of Protaspis grandis n. sp. 27. A transverse view of the multilayered cell wall showing pores through which extrusomes are presumably discharged (arrows) and a mitochondrion with tubular cristae (m) (Bar 5 15 mm). 28. High magnification transverse view of the cell wall showing seven distinct layers outside (above) the plasma membrane (pm): basal layer (bl), thick intermediate layer (il), vesicular layer (vl) embedded within the intermediate layer, and outer lamina (ol) squeezed between the intermediate layer and the coat (co). The coat contain three sub-layers: the darker deep coat (dc), the columnar mid-coat (mc), and the parallel superficial coat (sc) (Bar 5 0.1 mm). 29. Tangential view of the cell wall showing the regular spacing of the mid-coat (mc) and darker deep coat (dc) and expansion of the lighter zone within the outer lamina (ol). 30. Low magnification tangential view of the cell wall showing the central cytoplasm (cy), the coat (co), the deep coat (double arrowhead), the outer lamina (arrowhead) and the thick intermediate layer (il) pierced by pores (arrows) through which extrusomes are presumably discharged (Bar 5 0.5 mm). 31. Low magnification view of a battery of extrusomes oriented in two different directions: longitudinal (arrows) and transverse (arrowheads) (Bar 5 0.5 mm). 32. High magnification view of a membrane-bound extrusome (double arrowhead) showing its tip piercing the intermediate layer (il) through a pore (arrow) (Bar 5 0.2 mm). 336 J. EUKARYOT. MICROBIOL., VOL. 53, NO. 5, SEPTEMBER– OCTOBER 2006

Fig. 33. Illustrations comparing all of the described species within the genera Protaspis and Cryothecomonas showing the ventral furrow (if present), the orientation and insertion points of the heterodynamic flagella, relative position of the nucleus (n) within the cell, nuclear caps (arrows), anterior papillae (arrowhead), and anterior protrusions (double arrowheads). Warts are shown on the surface of Protaspis tegere, Protaspis verrucosa, Protaspis gemmifera, and Protaspis simplex. Pseudopodia are shown emerging from the ventral furrow in Protaspis metarhiza and Protaspis obovata. All illustrations are drawn approximately to scale. Redrawn after Skuja 1939 (Protaspis glans, Protaspis maior, Protaspis metarhiza), Skuja 1948 (Protaspis obovata), Lee 2001 (Protaspis tegere, Protaspis verrusosa, Protaspis gemmifera, Protaspis obliqua), Lee et al. 2005 (Protaspis simplex), Norris 1961 (Protaspis tanyopsis), Schnepf and Ku¨hn 2000 (Cryothecomonas longipes), Drebes et al. 1996 (Cryothecomonas aestivalis).

1996/97; Ku¨hn, Lange, and Medlin 2000). However, poor taxon among them. Gaining new sequences from microscopically iden- sampling of heterotrophic amoeboflagellates that potentially be- tified and characterized species (e.g. P. grandis) should provide long to the Cercozoa make it difficult to infer correct relationships new insights into the evolutionary history and diversity of HOPPENRATH & LEANDER—MORPHOLOGY AND PHYLOGENY OF PROTASPIS GRANDIS 337

Fig. 34. Gamma-corrected maximum likelihood tree (–ln L 5 8092.83, a 5 0.28, number of rate categories 5 4) inferred using the GTR model of substitution on an alignment of 54 small subunit (SSU) rDNA gene sequences and 1,006 sites. Numbers at the branches denote gamma-corrected bootstrap percentages using maximum likelihood—HKY (upper) and weighted neighbor-joining—GTR (middle). The lower number refers to Bayesian posterior probabilities—GTR. Black dots on branches denote bootstrap values and posterior probabilities of 100% in all analyses. The SSU rDNA sequence from Protaspis grandis n. sp. is highlighted in a black box and is closely related to Cryothecomonas longipes and other environmental sequences within the larger Cryomonadida clade. 338

Table 1. Comparison of morphological character states in described species of Protaspis and Cryothecomonas. 2006 OCTOBER SEPTEMBER– 5, NO. 53, VOL. MICROBIOL., EUKARYOT. J.

Characters P. obliqua P. grandis n. sp. P. glans P. gemmifera P. tegere P. verrucosa Flagella insertion Subapical Subapical, funnel Subapical Subapical Subapical Subapical Protrusion Protrusion No No No No Flagellar lengtha Anterior  0.5–0.75 cl  0.5 cl  0.5–1 Â cl  cl  1–2 Â cl  cl Posteriora  0.5–1.5 cl  cl 1.2–2 Â cl  1.3–3 Â cl  1.5–2.5 Â cl  1.3–2 Â cl Cell shape Round-oval Oval-rectangular Oval Round-oval Oblong-ovate Round-oval Cell surface Smooth Smooth Smooth Warty Smooth or warty Fine warts Cell flattening Dorso-ventral Dorso-ventral Dorso-ventral Dorso-ventral Dorso-ventral Dorso-ventral Cell size (mm) Length 08–32 32.5–55 12–30 08–17 14–25 09–22 Width 10–27 20–35 09–15 08–12 08–14 8.5–16 Anterior indentation Yes-left to median No No No No No Posterior indentation Yes No No No No No Nucleus position Anterior median Posterior-central Anterior or central lateral Anterior median Anterior, median or Anterior median right-hand side Nucleus shape Spherical Spherical Spherical Sphericalb SphericalNo/yes b DiscoidalNo/yes b CondensedNuclear cap chromosomes Unreported No Yes No Unreported No Unreported No/yes Unreported Unreported Nucleolus Yes Yes Unreported Unreported Unreported Unreported Ventral furrow/groove Posterior half, median Anterior half, median  Full cell length, median Indistinct  Full cell length, median  Full cell length, median Pseudopodia No Yes Yes Yes Yes Unreported Feeding mode Unreported Phagotrophy Phagotrophy Phagotrophy Phagotrophy Unreported Body scales Unreported No Unreported Unreported Unreported Unreported Flagella scales Unreported No Unreported Unreported Unreported Unreported Movement Gliding Gliding Gliding Gliding Gliding Gliding c only) Yes, multilayered Unreported Unreported Unreported Unreported Cell wall Yes (lm Extrusomes Unreported Yes Unreported Unreported Unreported Unreported Habitat Benthic Benthic Benthic, planktic Benthic Benthic, planktic Benthic, planktic OPNAH&LADRMRHLG N HLGN OF PHYLOGENY AND LEANDER—MORPHOLOGY & HOPPENRATH

Table 1. (Continued). Characters P. maior P. metarhiza P. obovata P. simplex P. tanyopsis Flagella insertion Subapical Subapical Subapical Subapical Subapical No No No No Protrusion Flagellar lengtha Anterior  cl  0.5–0.3 cl  cl  0.5–1 cl  1.2 cl Posteriora 2 Â cl  cl  cl 1–3 Â cl  1.3 cl Cell shape Oval Oval, posterior tip Ovoid (pear) Round-oval-ovoid Elongate Cell surface Smooth Smooth Smooth Fine warts in some Unreported Cell flattening Dorso-ventral Dorso-ventral Dorso-ventral Dorso-ventral Dorso-ventral Cell size (mm) Length 24–40 28–38 26–40 4.5–25 28–30 Width 16–30 16–27 17–25 02–10 09–11 Anterior indentation No No No, papilla No No Posterior indentation No No No, truncate No No Nucleus position Anterior median, Right lateral, Anterior Anterior, Anterior Antero-lateral Anterior–central Antero-lateral Nucleus shape Spherical Spherical-ovoid Spherical Ovoid Spherical Nuclear cap No No No Yes, in some cells No Condensed chromosomes Unreported Unreported Yes Unreported Unreported Nucleolus Yes Unreported Unreported Yes Unreported Ventral furrow/groove  cell length, oblique  cell length, left from median  cell length, straight or sigmoid Shallow, median  2/3 Cell length Pseudopodia Yes Yes Yes Not observed Not observed Feeding mode Phagotrophy Phagotrophy Phagotrophy Unreported Unreported

Body scales Unreported Unreported Unreported Unreported Unreported GRANDIS PROTASPIS Flagella scales Unreported Unreported Unreported Unreported Unreported Movement Gliding Gliding, swim Swim, rotational Gliding, wobbling Cell wall Unreported Unreported Unreported Unreported Unreported Extrusomes Unreported Unreported Unreported Unreported Unreported Habitat Benthic, planktic Planktic Planktic Benthic, planktic Planktic 339 340

Table 1. (Continued). 2006 OCTOBER SEPTEMBER– 5, NO. 53, VOL. MICROBIOL., EUKARYOT. J. Characters C. longipes C. aestivalis C. armigera C. inermis C. scybalophora C. vesiculata Flagellar insertion Apical & subapical, Apical, funnels, Apical, funnels, Apical, funnels, Apical, funnels, Apical, funnels, d Funnels, papilla Papilla Papilla Papilla Papilla Papilla Flagellar length Anterior 09–15 mm15mm 1–2 Â cell length 1–2 Â cell length Unreported Unreported Posterior 20–24 mm, fine hairs 25 mm25mm 1–2 Â cell length Unreported Unreported Unreported Heterodynamic Heterodynamic Isodynamic Isodynamic Unreported Unreported Cell shape Oval, kidney-shaped Oblong-oval Egg-shaped Egg-shaped Highly variable Elongated Cell surface Smooth Smooth Smooth Smooth With debris Protuberances Cell flattening Slightly No Yes Slightly No Slightly Cell size (mm): Length 09–14 09–12 12–32 10–15 09–14 10–15 Width 07–09 04–05 07–23 07–10 4.5–09 4–08 Nucleus position Anterior Anterior Anterior Anterior Anterior Anterior Nucleus shape Round, lobed Oval, lobed Round-oval Round-oval Round Round-oval Nuclear cap Unreported Unreported Unreported Unreported Unreported Unreported Condensed chromatin Yes Yes Yes Yes Yes Yes Nucleolus Yes Yes Yes Yes Yes Yes Furrow/groove Slit, ventral left side, 2/3 cell length Unreported 0.5–1 cell length Lateral, 0.5–1cell length Lateral Lateral Lateral Cytostome/slit Yes, anterior Yes Yes, posterio-lateral Yes, posterio-lateral Yes, posterio-lateral Yes Pseudopodia Yes Yes, posterior Yes Yes Yes Yes Feeding mode Phagotrophy Phagotrophy Phagotrophy Phagotrophy Phagotrophy Phagotrophy Body scales No No No No No No Flagella scales No No No No No No Movement Swim Swim, glide Unreported Unreported Unreported Unreported Cell wall/theca Yes, multilayered Yes, bilayered Yes, multilayered Yes, multilayered Yes, monolayered Yes, bilayered Extrusomes Yes No Yes No Yes Probably Muciferous bodies No No Yes Yes Yes Yes, many Habitat Planktic Planktic Planktic, sea ice Planktic, sea ice Planktic, sea ice Planktic, sea ice HOPPENRATH & LEANDER—MORPHOLOGY AND PHYLOGENY OF PROTASPIS GRANDIS 341 cercozoans (Ekelund, Daugbjerg, and Fredslund 2004; Ku¨hn, Chre´tiennot-Dinet, M.-J., Sournia, A., Ricard, M. & Billard, C. 1993. A Medlin, and Eller 2004). classification of the marine phytoplankton of the world from class to The Cercozoa was erected on the basis of molecular phyloge- genus. Phycologia, 32:159–179. netic data alone (Cavalier-Smith 1998a, b); no morphological Drebes, G., Ku¨hn, S. F., Gmelch, A. & Schnepf, E. 1996. Cryothecomonas feature characterizes the whole group and the taxonomic diag- aestivalis sp. nov., a colourless nanoflagellate feeding on the marine centric diatom Guinardia delicatula (Cleve) Hasle. Helgol. Wiss. nosis is unusually broad (Cavalier-Smith 1998a, p. 232). During Meeresunters., 50:497–515. the last 7 years, the identity and composition of the Cercozoa has Ekebom, J., Patterson, D. J. & Vrs, N. 1995/96. Heterotrophic flagellates been continuously modified (Bass and Cavalier-Smith 2004; from coral reef sediments (Great Barrier Reef, Australia). Arch. Pro- Cavalier-Smith and Chao 2003; Hoppenrath and Leander 2006). tistenkd., 146:251–272. The ‘‘undescribed cercozoan clade II’’ in Fig. 33 is the sister Ekelund, F. & Patterson, D. J. 1997. Some heterotrophic flagellates from a clade to the Cryomonadida and was shown to comprise ebriids cultivated garden soil in Australia. Arch. Protistenkd., 148:461–478. (Hoppenrath and Leander 2006). This resulted in the necessity to Ekelund, F., Daugbjerg, N. & Fredslund, L. 2004. Phylogeny of Hetero- amend the diagnosis for the Cercozoa to include taxa with internal mita, Cercomonas and Thaumatomonas based on SSU rDNA siliceous skeletons. Our observations of P. grandis require addi- sequences, including the description of Neocercomonas jutlandica sp. nov. gen. nov. Eur. J. Protistol., 40:119–135. tional amendments to the diagnosis of the clade in order to Felsenstein, J. 1993. PHYLIP (Phylogeny Inference Package). University accommodate taxa with a rigid protein layer outside of the plasma of Washington, Seattle. membrane (e.g. the cryomonads Protaspis and Cryothecomonas). Fensome, R. A., Taylor, F. J. R., Norris, G., Sarjeant, W. A. S., Wharton, These changes make the diagnosis for the clade even broader and D. I. & Williams, G. L. 1993. A classification of living and fossil make the prospect of defining the clade on morphological grounds dinoflagellates. Am. Mus. Nat. Hist., Micropaleontology special pub- alone even more remote. lication number 7:1–351. Hoppenrath, M. & Leander, B. S. 2006. Ebriid phylogeny and the expansion of the Cercozoa. Protist. (in press) Huelsenbeck, J. P. & Ronquist, F. 2001. MrBayes: Bayesian inference of ACKNOWLEDGMENTS phylogenetic trees. Bioinformatics, 17:754–755. Ku¨hn, S. F., Lange, M. & Medlin, L. K. 2000. Phylogenetic position of We wish to thank W. J. Lee and D. J. Patterson for discussions Cryothecomonas inferred from nuclear-encoded small subunit riboso- on a part of the manuscript and help with the literature. We would mal RNA. Protist, 151:337–345. like to acknowledge discussions with Ø. Moestrup about the Ku¨hn, S. F., Medlin, L. K. & Eller, G. 2004. Phylogenetic position of the taxonomic importance of flagellar insertions patterns and thank parasitoid nanoflagellate Pirsonia inferred from nuclear-encoded small G. Eller who generously provided a provisional alignment of subunit ribosomal DNA and a description of Pseudopirsonia n. gen. and small subunit rDNA sequences (‘‘Pseudopirsonia-alignment’’). Pseudopirsonia mucosa (Drebes) comb. nov. Protist, 155:143–156. This work was supported by a scholarship to M. Hoppenrath from Larsen, J. 1985. Algal studies of the Danish Wadden Sea. II. A taxonomic the Deutsche Forschungsgemeinschaft (grant Ho3267/1-1) and by study of psammobious dinoflagellates. Opera Bot., 79:14–37. grants to B. S. Leander from the National Science and Engineer- Larsen, J. & Patterson, D. J. 1990. Some flagellates (Protista) from tropical marine sediments. J. Nat. Hist., 24:801–937. ing Research Council of Canada (NSERC 283091-04) and the Leander, B. S., Clopton, R. E. & Keeling, P. J. 2003. Phylogeny of Canadian Institute for Advanced Research. B. S. Leander is a gregarines () as inferred from small subunit rDNA and Scholar of the Canadian Institute for Advanced Research, Pro- beta-tubulin. Int. J. Syst. Evol. Microbiol., 53:345–354. gram in Evolutionary Biology. Lee, W. J. 2001. Diversity and distribution of free-living benthic hetero- trophic flagellates in Botany Bay, Australia. Dissertation. University of Sydney, Sydney, Australia. 301 p. Lee, W. J. & Patterson, D. J. 2000. Heterotrophic flagellates (Protista) LITERATURE CITED from marine sediments of Botany Bay, Australia. J. Nat. Hist., 34: Adl, S. M., Simpson, A. G. B., Farmer, M. A., Andersen, R. A., Anderson, 483–562. O. R., Barta, J. R., Bowser, S. S., Brugerolle, G., Fensome, R. A., Lee, W. J., Brandt, S. M., Vrs, N. & Patterson, D. J. 2003. Darwin’s Fredericq, S., James, T. Y., Karpov, S., Kugrens, P., Krug, J., Lane, C. heterotrophic flagellates. Ophelia, 57:63–98. R., Lewis, L. A., Lodge, J., Lynn, D. H., Mann, D. G., McCourt, R. M., Lee, W. J., Simpson, A. G. B. & Patterson, D. J. 2005. Free-living Mendoza, L., Moestrup, Ø., Mozley-Standridge, S. E., Nerad, T. S., heterotrophic flagellates from freshwater sites in Tasmania (Australia), Shearer, C. A., Smirnov, A. V., Spiegel, F. W. & Taylor, F. J. R. 2005. a field survey. Acta Protozool., 44:321–350. The new high level classification of eukaryotes with emphasis on the Loeblich, A. R. III. 1969. The amphiesma or dinoflagellate cell covering. taxonomy of Protists. J. Eukaryot. Microbiol., 52:399–451. Proc. N. Am. Pal. Conv., Part G:867–929. Auer, B. & Arndt, H. 2001. Taxonomic composition and biomass of Loeblich, A. R. Jr. & Loeblich, A. R. III. 1966. Index to the genera, subgenera, heterotrophic flagellates in relation to lake trophy and season. Fresh- and sections of the Pyrrhophyta. Stud. Trop. Oceanogr., 3:1–94. water Biol., 46:959–972. Maddison, D. R. & Maddison, W. P. 2000. Sinauer Associates, Inc., Bass, D. & Cavalier-Smith, T. 2004. Phylum-specific environmental DNA MacClade Sunderland, MA. analysis reveals remarkably high global biodiversity of Cercozoa (Pro- Mylnikov, A. P. & Karpov, S. A. 2004. Review of diversity and taxonomy tozoa). Int. J. Syst. Evol. Microbiol., 54:2393–2402. of cercomonads. , 3:201–217. Bruno, W. J., Socci, N. D. & Halpern, A. L. 2000. Weighted neighbor Norris, R. E. 1961. Observations on phytoplankton organisms collected on joining: a likelihood-based approach to distance-based phylogeny re- the N.Z.O.I. Pacific cruise, Sept. 1958. N.Z. J. Sci., 4:162–188. construction. Mol. Biol. Evol., 17:189–197. Patterson, D. J. & Zo¨lffel, M. 1991. Heterotrophic flagellates of uncertain Cavalier-Smith, T. 1998a. A revised six- system of life. Biol. taxonomic position. In: Patterson, D. J. & Larsen, J. (ed.), The Biology Rev., 73:203–266. of Free-living Herotrophic . Systematics Association Spe- Cavalier-Smith, T. 1998b. Neomonada and the origin of animals and cial. Vol. 45. Clarendon Press, Oxford. p. 427–475. fungi. In: Coombs, G. H., Vickermann, K., Sleigh, M. A. & Warren, A. Patterson, D. J., Nygaard, K., Steinberg, G. & Turley, C. M. 1993. (ed.), Evolutionary Relationships Among . Chapman & Hall, Heterotrophic flagellates and other protists associated with oceanic London. p. 375–407. detritus throughout the water column in the mid-North Atlantic. Cavalier-Smith, T. & Chao, E. E. 1996/97. Sarcomonad ribosomal RNA J. Mar. Biol. Assoc. UK, 73:67–95. sequences, rhizopod phylogeny, and the origin of euglyphid amoebae. Patterson, D. J., Vrs, N., Simpson, A. G. B. & O’Kelly, C. 2002. Residual Arch. Protistenkd., 147:227–236. free-living and predatory heterotrophic flagellates. In: Lee, J. J., Lee- Cavalier-Smith, T. & Chao, E. E. 2003. Phylogeny and classification of dale, G. F. & Bradbury, P. (ed.), The Illustrated Guide to the Protozoa. phylum Cercozoa (Protozoa). Protist, 154:341–358. 2nd ed. Society of Protozoologists, Lawrence, KS. p. 1302–1328. 342 J. EUKARYOT. MICROBIOL., VOL. 53, NO. 5, SEPTEMBER– OCTOBER 2006

Posada, D. & Crandall, K. A. 1998. MODELTEST: testing the model of Strimmer, K. & Von Haeseler, A. 1996. Quartet puzzling: a quartet DNA substitution. Bioinformatics, 14:817–818. maximum likelihood method for reconstructing tree topologies. Mol. Schnepf, E. & Ku¨hn, S. F. 2000. Food uptake and fine structure of Biol. Evol., 13:964–969. Cryothecomonas longipes sp. nov., a marine nanoflagellate incertae Swofford, D. L. 1999. Phylogenetic Analysis Using Parsimony (and Other sedis feeding phagotrophically on large diatoms. Helgol. Mar. Res., Methods) PAUPÃ 40. Sinauer Associates Inc, Sunderland, MA. 54:1–32. Thomsen, H. A., Buck, K. R., Bolt, P. A. & Garrison, D. L. 1991. Fine Silva, P. C. 1980. Names of Classes and Families of Living . Bohn, structure and biology of Cryothecomonas gen. nov. (Protista incertae Scheltema & Holkema, Utrecht. sedis) from the ice biota. Can. J. Zool., 69:1048–1070. Skuja, H. 1939. Beitrag zur Algenflora Lettlands II. Acta horti botanici Tong, S. M., Nygaard, K., Bernard, C., Vrs, N. & Patterson, D. J. 1998. Universitatis latviensis, 11/12:41–169. Heterotrophic flagellates from the water column in Port Jackson, Skuja, H. 1948. Taxonomie des Phytoplanktons einiger Seen in Uppland, Sydney, Australia. Europ. J. Protistol., 34:162–194. Schweden. Symb. Bot. Ups., 9:1–399. Uhlig, G. 1964. Eine einfache methode zur extraktion der vagilen, meso- Sournia, A. 1973. Catalogue des espe`ces et taxons infraspe´cifiques de psammalen Mikrofauna. Helgol. Wiss. Meeresunters., 11:178–185. Dinoflagelle´s marins actuels publie´s depuis la re´vision de J. Schiller. I. Vrs, N. 1992. Heterotrophic amoebae, flagellates and from the Dinoflagelle´s libres. Nova Hedwigia, 48:1–92. Tva¨rminne area, Gulf of Finland, in 1988–1990. Ophelia, 36:1–109. Sournia, A. 1978. Catalogue des espe`ces et taxons infraspe´cifiques de Vrs, N. 1993. Heterotrophic amoebae, flagellates and heliozoa from Dinoflagelle´s marins actuels publie´s depuis la re´vision de J. Schiller. III. Arctic marine waters (North West Territories, Canada and West Green- (Comple´ment). Rev. Algol., 13:3–40. land). Polar Biol., 13:113–126. Sournia, A. 1986. Atlas du phytoplankton marin. Vol I: Introduction, Zolan, M. E. & Pukkila, P. J. 1986. Inheritance of DNA methylation in Cyanophyce´es, Dictyochophyce´es, Dinophyce´es et Raphidophyce´es. Corprinus cinereus. Mol. Cell. Biol., 6:195–200. E´ ditions du CNRS, Paris. Sournia, A. 1993. Catalogue des espe`ces et taxons infraspe´cifiques de Dinoflagelle´s marins actuels publie´s depuis la re´vision de J. Schiller. VI. (Comple´ment). Cryptogam. Algol., 14:133–144. Received: 12/04/05, 03/30/06, 04/03/06; accepted: 04/03/06