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pseudopods formed out of the furrow (Skuja 1939). Skuja (1948) described a fourth species, Protaspis obovata. The large

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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.

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HE genus Protaspis was described by Skuja (1939), with the type species Protaspis glans and two additional species, Protaspis maior and Protaspis metarhiza (Skuja 1939). Skuja (1939) erected the new family Protaspidaceae for his new genus and classified it tentatively within the euglenids, because of the strong continuous ‘‘periplast,’’ the ventral longitudinal furrow, the two heterodynamic flagella, the large nucleus with nucleolus and the paramylon-like reserve product. The flagella insert in the anterior part of the ventral furrow and are clearly separated from each other. The movement is by gliding and food uptake is by pseudopods formed out of the furrow (Skuja 1939). Skuja (1948) described a fourth species, Protaspis obovata. The large nucleus of this species had the characteristic morphology of a dinoflagellate nucleus (dinokaryon), which led Skuja (1948) to reclassify Protaspis within the Pyrrophyta (dinoflagellates). This unusual combination of characters led Skuja (1948) to entertain the possibility that protaspids might occupy an intermediate phylogenetic position between euglenids and dinoflagellates. From the time of Skuja’s work to the early 1990s, Protaspis was consistently treated as a dinoflagellate taxon (Chre´tiennotDinet et al. 1993; Loeblich 1969; Loeblich and Loeblich 1966; Silva 1980; Sournia 1973, 1978, 1986, 1993). Fensome et al. (1993) excluded Protaspis from the division Dinoflagellata and stated that it is a problematic genus, possibly of euglenid affinity. However, at about that same time, Protaspis was also tentatively classified as belonging to the Thaumatomastigaceae/Thaumatomastigidae (Larsen and Patterson 1990; Patterson et al. 2002; Patterson and Zo¨lffel 1991). Mylnikov and Karpov (2004) argued that Protaspis should be transferred into the Cercomonadida because protaspids do not have a flagellar pocket and unlike thaumatomonads, do not have body scales. This opinion is reflected in the latest higher-level classification of eukaryotes, where Protaspis is classified in the Cercomonadida in the ‘‘Family’’ Heteromitidae (Adl et al. 2005).

Corresponding Author: M. Hoppenrath, Canadian Institute for Advanced Research, Program in Evolutionary Biology, Departments of Botany and Zoology, University of British Columbia, Vancouver, BC, Canada V6T 1Z4—Telephone number: 604-822-4892; FAX number: 604-822-6089; e-mail: [email protected]

Protaspis species occur in marine benthic habitats, freshwater, and marine plankton communities, and soil (Auer and Arndt 2001; Ekelund and Patterson 1997; Ekebom, Patterson, and Vrs 1995/ 96; Larsen 1985; Larsen and Patterson 1990; Lee and Patterson 2000; Lee et al. 2003, 2005; Norris 1961; Patterson et al. 1993; Skuja 1939, 1948; Tong et al. 1998; Vrs 1992, 1993). Currently, the genus contains 10 species: P. glans Skuja 1939, P. maior Skuja 1939, P. metarhiza Skuja 1939, P. obovata Skuja 1948, Protaspis tanyopsis Norris 1961, Protaspis gemmifera Larsen and Patterson 1990, Protaspis obliqua Larsen and Patterson 1990, Protaspis tegere Larsen and Patterson 1990, Protaspis verrucosa Larsen and Patterson 1990, and Protaspis simplex Vrs 1992. The distinguishing features among some of these species are not clear, and it is likely that some Protaspis species will prove to be conspecific (for detailed discussion in Lee 2001). The unresolved phylogenetic position of Protaspis and the potential affiliation to dinoflagellates or euglenids motivated us to investigate the phylogeny of a new species, Protaspis grandis n. sp., on the basis of ultrastructural and small subunit (SSU) rDNA sequence data. MATERIALS AND METHODS Collection of organisms. Samples were collected with a spoon during low tide at Centennial Beach, Boundary Bay, BC, Canada. The salinity of the water is about 30–33 psu. The sand samples were transported directly to the laboratory and the flagellates were separated from the sand by extraction through a 45-mm filter using melting seawater-ice (Uhlig 1964). The flagellates accumulated in a Petri dish beneath the filter and were then identified with an inverted-microscope at 40  –250  magnification. Cells were isolated by micropipetting and used directly (not from culture) for the preparations described below. Light microscopy. Cells were observed directly and micromanipulated with a Leica DMIL inverted microscope. For differential interference contrast light microscopy, micropipetted cells were placed on a glass specimen slide and covered with a cover slip. Images were produced with a Zeiss Axioplan 2 imaging microscope connected to a Leica DC500 color digital camera. Scanning electron microscopy. A mixed-extraction sample was fixed overnight with two drops of acidic Lugol’s solution. Cells were transferred onto a 5-mm polycarbonate membrane filter

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(Corning Separations Div., Acton, MA), washed with distilled water, dehydrated with a graded series of ethanol and critical point dried with CO2. Filters were mounted on stubs, sputter-coated with gold and viewed under a Hitachi S4700 Scanning Electron Microscope (SEM). Some SEM images were presented on a black background using Adobe Photoshop 6.0 (Adobe Systems, San Jose, CA). Transmission electron microscopy. Cells were concentrated in a microfuge tube by micropipetting and slow centrifugation. The pellet of cells was prefixed with 2% (v/v) glutaraldehyde in seawater at 4 1C for 30 min. Cells were washed twice in filtered seawater (30–35 psu) before post-fixation in 1% (w/v) OsO4 in seawater for 30 min at room temperature. Cells were dehydrated through a graded series of ethanol, infiltrated with acetone–resin mixtures (pure acetone, 2:1, 1:1, 1:2, pure resin), and embedded in pure resin (Epon 812). The block was polymerized at 60 1C and sectioned with a diamond knife on a Leica Ultracut UltraMicrotome. Thin sections were post-stained with uranyl acetate and lead citrate and viewed under a Hitachi H7600 Transmission Electron Microscope. DNA extraction, amplification, cloning and squencing. Forty individually isolated (uncultured) cells were washed three times in filtered (eukaryote-free) seawater. Genomic DNA was extracted from the 40-cell preparation using a standard hexadecyltrimethylammonium bromide (CTAB) extraction protocol (Zolan and Pukkila 1986). The PCR amplification protocol using universal eukaryotic primers reported previously (Leander, Clopton, and Keeling 2003) consisted of an initial denaturing period (95 1C for 2 min); 35 cycles of denaturing (92 1C for 45 s), annealing (48 1C for 45 s), and extension (72 1C for 1.5 min); and a final extension period (72 1C for 5 min). Polymerase chain reaction products corresponding to the expected size were gel isolated and cloned into the pCR2.1 using the TOPO TA cloning kit (Invitrogen, Burlington, ON, Canada). Two clones were sequenced with ABI big-dye reaction mix using the vector primers and internal primers oriented in both directions. Phylogenetic analyses. The Protaspis SSU rDNA sequence 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 gaps were excluded from the alignments before phylogenetic analysis. The a shape parameters were estimated from the data using HKY under a g distribution plus invariable sites model and four rate categories (a 5 0.28, Ti/Tv 5 1.38, fraction of invariable sites 5 0.08). A gamma-corrected ML tree (analyzed using the parameters listed above) was constructed with PAUP 4.0 using the general time reversible (GTR) model for base substitutions (Posada and Crandall 1998; Swofford 1999). Gamma corrected ML tree topologies found with HKY and GTR were identical. Maximum likelihood bootstrap analyses were performed in PAUP 4.0 (Swofford 1999) on 100 re-sampled datasets under an HKY model using the a shape parameter, proportion of invariable sites and transition/transversion ratio (Ti/Tv) estimated from the original dataset. Maximum likelihood distances for the SSU rDNA dataset were calculated with TREE-PUZZLE 5.0 using the GTR substitution matrix (Strimmer and Von Haeseler 1996). A distance tree was constructed with weighted neighbor joining (WNJ) using Weighbor (Bruno, Socci, and Halpern 2000). Five-hundred bootstrap datasets were generated with SEQBOOT (Felsenstein 1993). Respective distances were calculated with the shell script ‘‘puzzleboot’’ (M. Holder and A. Roger, www.tree-puzzle.de) using the a shape parameter and proportion of invariable sites estimated from the original dataset and analyzed with Weighbor.

We also examined the dataset with Bayesian analysis using the program MrBayes 3.0 (Huelsenbeck and Ronquist 2001). The program was set to operate with GTR, a g distribution and four MCMC chains starting from a random tree (default temperature 5 0.2). A total of 2,000,000 generations were calculated with trees sampled every 100 generations and with a prior burn-in of 200,000 generations (2,000 sampled trees were discarded). A majority rule consensus tree was constructed from 16,000 postburn-in trees with PAUP 4.0. Posterior probabilities correspond to the frequency at which a given node is found in the post-burn-in trees. GenBank accession numbers. Allas diplophysa (AF411262), Allas sp. (AF411263), Bigelowiella natans (AF054832), Cercomonas longicauda (AF101052), Cercomonas sp. (U42448), Cercozoa sp. WHO1 (AF411273), Chlorarachnion reptans (U03477), Cryothecomonas aestivalis (AF290541), Cryothecomonas longipes (AF290540), Euglypha rotunda (AJ418784), Gromia oviformis (AJ457813), Heteromita globosa (U42447), Lotharella globosa (AF076169), Massisteria marina (AF174372), Paulinella chromatophora (X81811), Phagomyxa bellerochea (AF310903), Polymyxa betae (AF310902), P. grandis n. sp. (DQ303924), Pseudodifflugia cf. gracilis (AJ418794), Pseudopirsonia mucosa (AJ561116), Thaumatomastix sp. (AF411261), Thaumatomonas seravini (AF411259), Thaumatomonas sp. (U42446), Thaumatomonas sp. (SA) (AF411260), uncultured cercozoan (AF372764), uncultured cercozoan (AY180012), uncultured cercozoan (AY180035), uncultured cercozoan (AY620274), uncultured cercozoan (AY620276), uncultured cercozoan (AY620277), uncultured cercozoan (AY620279–AY620281), uncultured cercozoan (AY620294), uncultured cercozoan (AY620295), uncultured cercozoan (AY620300), uncultured cercozoan (AY620314), uncultured cercozoan (AY620316), uncultured cercozoan (AY620320– AY620323), uncultured cercozoan (AY620340), uncultured cercozoan (AY620348–AY620353), uncultured cercozoan (AY620355), uncultured cercozoan (AY620357), uncultured eukaryote TAGIRI-2 (AB191410), uncultured eukaryote (AJ130856), uncultured eukaryote (AY082998).

RESULTS Cell shape and flagella. The flagellated stage in the life cycle of P. grandis was investigated. Plasmodial and cyst stages were not observed. Cells were 32.5–55.0 mm long, 20.0–35.0 mm wide (n 5 21), dorso-ventrally flattened and shaped like elongated ovals with parallel lateral sides (Fig. 1–4). The ventral side of the cell was concave and the dorsal side was slightly convex (Fig. 5). Two thin, heterodynamic flagella emerged from separate subapical depressions on the ventral side of the cell (Fig. 4, 11). The anterior flagellum was about half a cell length, and the trailing flagellum was about one cell length. A protrusion emerging from the right furrow border pointed to the left and separated the insertion depressions of the flagella (Fig. 1, 4, 11, 15). The posterior flagellum inserted below the protrusion and was positioned slightly to the left. A narrow ventral furrow is positioned medially and extends about one-third of the cell length posteriorly from the insertion point of the posterior flagellum and towards the posterior end (Fig. 1, 3, 4, 11). The flagella were anchored within flagellar pits and emerged through funnels with a sharp outer margin and ring-shaped basal depression (Fig. 16). Transmission electron micrographs through this region of the cell contained a structure that lacked the typical 912 organization of microtubules and was composed of material arranged in concentric rings of different density (Fig. 17). This morphology is consistent with both a flagellum, in transverse view, as seen in C. aestivalis (Drebes et al. 1996, Fig. 23) and an extrusome, in transverse view.

HOPPENRATH & LEANDER—MORPHOLOGY AND PHYLOGENY OF PROTASPIS GRANDIS

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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 granular appearance was situated in the posterior half of the cell and midway between the lateral margins (Fig. 2, 4, 5). The nucleus was never observed in a lateral position or in the anterior half of the cell. Nuclear caps were not observed. The nucleus contained a large nucleolus and permanently condensed chromosomes distributed evenly (Fig. 12, 13). Some of our micrographs showed a convoluted cleft in the anterior side of the nucleus (Fig. 13). The cytoplasm was generally colorless and contained food vacuoles, masses of small vesicles/granules, and often some larger colored particles (e.g. yellow, orange, red, or brown) (Fig. 1–4, 12, 13). The ultrastructure of the large colored particles was uniform and often darkly stained suggesting that they were globules of lipids (Fig. 12, 13). Mitochondria were evenly distributed throughout the cytoplasm and had tubular-shaped cristae (Fig. 14). The cytoplasm also contained elongated Golgi bodies composed of stacks of about six to nine cisternae (Fig. 20). Ventral furrow and pseudopodia. In some cells, relatively long and thin pseudopodia emerged from the ventral furrow and spread over the substrate surface (not shown). An unidentified Protaspis cell with typical extruded pseudopodia is shown in Fig. 10. Pseudopodia were evident in our P. grandis samples prepared for SEM and TEM as spherical globules located near the ventral furrow (Fig. 18, 19). The ventral furrow was observed in three different states: closed (Fig. 21), open (Fig. 22), and protruding (Fig. 23). A distinctive slit was located inside the ventral furrow, from which the pseudopodia emerged (Fig. 19, 22–24). The pseudopodia were bordered by the plasma membrane, lacked a cell wall, and were rich in mitochondria (Fig. 25). Before cell

division, the flagellar apparatus doubled (Fig. 26). Although cell division was not observed during this study, the pattern of flagellar replication indicated that longitudinal division most likely occurs along a longitudinal cleavage furrow, which has also been described for other species in the genus. Cell wall. The cell surface was smooth and reinforced with a thickened cell wall comprised of at least seven different layers of material having different thicknesses, densities, and architectures (Fig. 2, 3, 11, 12, 15, 27, 28). The multilayered wall was secreted outside of the plasma membrane (Fig. 28). An amorphous ‘‘basal layer’’ about 150 nm thick was positioned directly above the plasma membrane and was overlain by a broad ‘‘intermediate layer’’ about 400 nm thick (Fig. 28). A ‘‘vesicular layer’’ (50 nm thick) interrupted the relatively homogeneous intermediate layer near its superficial margin (Fig. 28). A thin electron-dense ‘‘outer lamina’’ was squeezed between the intermediate layer and a sealing ‘‘coat’’ consisting of three ridged layers: a darker ‘‘deep coat,’’ a columnar ‘‘mid-coat,’’ and a parallel ‘‘superficial coat’’ (Fig. 27, 28). In tangential section, the regular spacing of the ridges of the coat were easily discernable (Fig. 29). Cylindrical pores pierced the basal and intermediate layers and permitted extrusomes to be discharged through the cell wall (Fig. 27, 30– 32). Pores were never observed piercing the tri-layered coat (Fig. 27). Batteries of extrusomes oriented in the same direction were usually concentrated in clusters (Fig. 31), and each extrusome was located within a membrane-bound vesicle (Fig. 32). Molecular phylogeny. We sequenced the SSU rRNA gene from a multi-specimen sample of P. grandis collected in September 2004. Basic local alignment search tool results robustly placed

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the sequence together with C. longipes and several related environmental sequences, all of which belonged to the ‘‘Cercozoa.’’ We created a 54-taxon alignment (1,006 unambiguous sites) consisting of ingroup cercozoans and representative sequences from plasmodiophorids, chlorarachniophytes, and Gromia (Fig. 33). Protaspis grandis, C. longipes, and three environmental sequences (AY620323, AY620340, and AY620352) branched together with strong support from both bootstrap statistics and Bayesian posterior probabilities (Fig. 33). This subclade fell within a much larger clade containing C. aestivalis and several environmental sequences, all of which formed the well-supported Cryomonadida clade. Interestingly, C. longipes branched much more closely to P. grandis than to C. aestivalis (Fig. 33). Nonetheless, the Cryomonadida clade formed the sister group to a sequence clade of unknown identity, namely ‘‘undescribed cecozoan clade II’’ and was only distantly related to the Thaumatomonadida (Fig. 33). DISCUSSION Comparative morphology. The overall light-microscopical appearance of the species described here conforms with the characteristic features for the genus Protaspis: heterodynamic flagella insert subapically within separate depressions on the ventral surface of the cell, movement is by substrate-mediated gliding, feeding is by means of pseudopodia that emerge from a ventral furrow and the granularity of the nucleus is clearly visible. Protaspis grandis n. sp. is larger than all other described species in the genus, but its smallest size overlaps with the largest size range of the larger species, P. maior, P. metarhiza, and P. obovata (Table 1). Protaspis grandis n. sp. is clearly distinguishable from these species and nearly all other Protaspis species by having an anterior protrusion that separates the flagellar insertion points and an obviously thick cell wall (Fig. 34). Only P. tanyopsis and P. obliqua have a similar protrusion (Larsen and Patterson 1990; Lee and Patterson 2000; Lee, Simpson, and Patterson 2005; Norris 1961). Protaspis tanyopsis also has a similar shape to P. grandis but is smaller, only slightly flattened dorso-ventrally, and lacks a thickened cell wall. Moreover, P. tanyopsis is longer than P. grandis relative to the cell width and the nucleus is positioned anteriorly rather than posteriorly (Norris 1961; Table 1 and Fig. 34). Norris (1961) described P. tanyopsis as having a small lobe near the anterior end of the groove margin but did not specify which margin, the left or right side of the groove. Although we remain doubtful, his drawing suggests that the lobe/protrusion is part of the left-hand groove margin (Fig. 34). By contrast, the anterior protrusion of P. grandis and P. obliqua emerges from the right-hand furrow margin. Like P. grandis, P. obliqua also has a thickened cell wall (Larsen and Patterson 1990; Lee 2001; Lee and Patterson 2000; Lee et al. 2005). However, the cells of P. obliqua are significantly smaller than P. grandis, are round to oval rather than elongated, and have a posterior indentation that gives

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the cell an asymmetrical appearance (Fig. 34). Moreover, the furrow of P. obliqua is in the posterior half of the cell and the nucleus is positioned in the anterior half, which stands in contrast to the anterior furrow and posterior nucleus position in P. grandis (Larsen and Patterson 1990, 2000; Table 1; Lee 2001 and Fig. 34). These morphological characters clearly distinguish P. obliqua from P. grandis, and we were always able to distinguish these two species in samples in which both of these species co-occurred (Fig. 1–9). The P. obliqua cells were 27.5– 35.0 mm long and 25.0–32.5 mm wide (n 5 10), which is slightly larger than that reported in the literature (Table 1). No intermediate morphologies between P. obliqua and P. grandis were observed in our samples. Therefore, we establish Protaspis grandis n. sp. and provide the following species description. Taxon Description Cercozoa Cavalier-Smith 1998, emend. Adl et al. 2005 Cercomonadida Poche 1913, emend. Vickerman 1983, emend. Mylnikov 1986 Heteromitidae Kent 1880, emend. Mylnikov 1990, emend. Mylnikov and Karpov 2004 Protaspis Skuja 1939 Protaspis grandis Hoppenrath et Leander n. sp. Diagnosis. Cells shaped as elongated ovals with parallel lateral sides, dorsoventrally flattened, 32.5–55.0 mm long, 20.0–35.0 mm wide. Two heterodynamic flagella, inserted subapically, separated by a protrusion. Flagellar pits with funnel. Ventral longitudinal furrow in the anterior half of cell. Nucleus in the posterior half of cell, with nucleolus and permanently condensed chromosomes. Pseudopodia emerge through a slit within the ventral furrow. Thickened cell wall outside of the plasma membrane consisting of seven layers: basal layer, intermediate layer, vesicular layer, outer lamina, deep coat, mid-coat, and superficial coat. Basal layer and intermediate layer pierced by pores for the discharge of extrusomes. Holotype/type micrograph. Fig. 4. Type locality. Tidal sand-flat at Centennial Beach, Boundary Bay, BC, Canada. The species was observed with higher abundance in September 2004 and 2005, but also in October 2004, March, April, June, and August 2005 in low cell numbers. Habitat. Marine. Etymology for the specific epithet. Refers to the large cell size relative to all other known species within the genus. Which characters are useful for the recognition of different species? Vrs (1992) stated that the species are ‘‘. . . distinguished by size and shape, the length and path of the groove, and the presence or absence of (1) a nuclear cap, (2) a protrusion separating the flagella, (3) rod-shaped bodies of reserve material [and] . . . 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 (lefthand 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 vacuole (fv), and darkly stained lipid globules (l) (Bar 5 2 mm). 14. TEM showing a typical double membrane-bound mitochondrion 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).

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and a warty cell surface’’ (p. 91). It has been noticed that the size range of Protaspis species was often expanded when further observations of a species were made in different habitats (e.g. Lee 2001; Lee and Patterson 2000). Most of the Protaspis species show considerable overlap in their size ranges (Table 1), and therefore cell size is only of value when the differences are relatively discrete and are used in connection with other features. The flattened cell shape of most of Protaspis species is very similar (round to oval to ovoid; Table 1 and Fig. 34), and most likely there is a limited degree of variation (Table 1) depending on the age of the cell, feeding stages and cell cycle stages (e.g. P. obovata in Skuja 1948). The cell shape must differ significantly in order to be a distinguishing character by itself (e.g. round in contrast to elongated oval). The morphology of the ventral furrow seems to be a useful character, even if the path and visibility varies in older cells (e.g. P. maior in Skuja 1939). The length of the furrow in relation to the cell length, the width (e.g. broad versus widening at the ends versus narrow), and the position (e.g. indistinct versus whole cell length versus mainly anterior versus mainly posterior) of the furrow appear to be reasonable diagnostic characters (Table 1). The presence or absence of nuclear caps is not a consistent feature or has not been recorded reliably (Tong et al. 1998); P. gemmifera, P. tegere, and P. verrucosa were all described both with and without them (Larsen and Patterson 1990; Lee 2001; Lee and Patterson 2000; Patterson et al. 1993; Tong et al. 1998). The anterior protrusion separating the flagella is still regarded as a consistent species character. Special cell inclusions like conspicuous reserve material (e.g. rod-shaped in P. gemmifera) are no longer used as a characteristic feature (Lee 2001; Lee and Patterson 2000). The presence or absence of warts on the cell surface is not a constant character or not reliably observed (e.g. P. gemmifera, P. tegere and P. simplex; Ekelund and Patterson 1997; Ekebom et al. 1995/96; Larsen and Patterson 1990; Lee 2001; Lee and Patterson 2000; Lee et al. 2005; Vrs 1992). The position of the nucleus is nearly the same in all Protaspis species (Table 1 and Fig. 34) and varies to a certain extent in P. metarhiza and P. glans depending on the age of the cells (Larsen 1985; Larsen and Patterson 1990; Skuja 1939). Protaspis grandis is the only species described so far with the nucleus positioned in the posterior half of the cell. The thickness of the cell wall is a distinguishing feature, with only P. obliqua and P. grandis having a thick one that is easily visible with light microscopy (Larsen and Patterson 1990; Lee and Patterson 2000; Fig. 1–9). A posterior (and anterior?) indentation seems to be a special feature, so far only described for P. obliqua (Larsen and Patterson 1990; Lee 2001; Lee and Patterson 2000). Moreover, there is large and overlapping range for flagellar lengths within a species (Table 1), making it useless for species identification. The mode of flagellar movement could be of interest, but this behavioral character has not been sufficiently documented for all of the described species. The possibility of different cell behaviors during different feeding and cell cycle stages would make the use of these characteristics for species separation challenging.

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Pseudopodia are also difficult to observe and the morphological details associated with these structures are not available for all the species, which questions the taxonomic value of these features. Therefore, in our view, characters like anterior protrusions, anterior or posterior indentations, furrow morphology, and the relative thickness of the cell wall are the best diagnostic features currently available. In addition, the position of the nucleus within the cell (e.g. anterior versus posterior) and cell shape (round versus elongated) used in connection with discrete differences in the range of cell sizes appear to be practical ‘‘combinationcharacters’’ (Fig. 34). After taking the above discussion about the usefulness of certain characters into account, there is the strong possibility that P. glans, P. maior, P. metarhiza, and P. tegere are actually conspecific. Lee (2001) also discussed this possibility and also included P. obovata to the list. We think that the apex morphology with a papilla-like structure in combination with the truncated antapex characterizes P. obovata, and we therefore do not regard it as synonymous. Protaspis gemmifera and P. simplex are probably also synonymous. Perhaps, the conspecificity of these taxa should be demonstrated with molecular methods before taxonomic changes are proposed. Nonetheless, it is probable that the genus will ultimately be reduced from eleven to seven species: P. glans, P. obovata, P. tanyopsis, P. gemmifera, P. verrucosa, P. obliqua, and P. grandis. It appears that other species of Protaspis have been observed but not formally described. For instance, Sournia (1986) reported a light micrograph of two cells that was referred to as Protaspis of unknown identity (p. 143, Fig. 51). Although the organization of flagellar insertions and pseudopodia were not visible, the overall morphology of this cell strongly suggests that it is a Protaspis species with a thick cortex, anterior nucleus and long ventral furrow. Our phylogenetic analyses of P. grandis demonstrates a close relationship between this species and C. longipes Schnepf et Ku¨hn 2000 (Fig. 33). This result is consistent with ultrastructural similarities between the two species. Like P. grandis, C. longipes has a multilayered cell wall, a nucleus with a conspicuous nucleolus and condensed chromosomes, extrusomes of the same morphology, pseudopodia that protrude through a preformed slit and flagella that sit within separated flagellar pits and emerge through distinctive funnels (Schnepf and Ku¨hn 2000; Fig. 12–32). A multilayered cell wall, a nucleus with distinct areas of condensed chromatin and flagella emerging through funnels are ultrastructural characters that have been used to diagnose the genus Cryothecomonas (Thomsen et al. 1991; Table 1). Nonetheless, there are clear ultrastructural differences between P. grandis and C. longipes, such as the organization of the flagellar insertion points, the abundance and distribution of condensed chromosomes, the presence of a ventral furrow and the relative thicknesses of cell wall layers. The anterior flagellum is inserted apically and the posterior flagellum inserted subapically in C. longipes (Schnepf and Ku¨hn 2000), whereas both flagella are inserted subapically in all Protaspis species (Table 1 and Fig. 34). 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).

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reflect different swimming modes associated with the benthic and planktonic lifestyles of most Protaspis and Cryothecomonas species, respectively. The nucleus of C. longipes is lobed, with a prominent nucleolus surrounded by heterochromatin aggregated near the periphery of the nucleus (Schnepf and Ku¨hn 2000), whereas the heterochromatin in P. grandis is more abundant and evenly distributed in a non-lobed nucleus. Unlike P. grandis, the ventral slit in C. longipes is not situated in a ventral furrow (Schnepf and Ku¨hn 2000). Key differences in the multilayered cell wall of P. grandis, when compared with C. longipes are as follows: (1) the coat is more stratified containing not only a columnar mid-coat layer (like C. longipes) but also a deep coat layer and a parallel superficial coat layer; (2) the intermediate layer is significantly thicker and contains a vesicular layer embedded within it (putatively homologous to the ‘‘compact layer’’ of C. longipes); and (3) the basal layer is much thicker (Fig. 12, 27–30). Although our naming scheme is consistent with the descriptions presented for C. longipes (Schnepf and Ku¨hn 2000), we were unable to rule out the possibility that the ‘‘outer lamina’’ in P. grandis actually represents the plasma membrane (Fig. 27–30). These relatively modest differences in the basic characters between these two species raise the question of whether P. grandis and C. longipes should be classified within two different genera. Moreover, is it sensible for us to generalize these observations to all members of both genera? If so, is it warranted to transfer P. grandis and related Protaspis species into the genus Cryothecomonas or, conversely, to transfer C. longipes into the genus Protaspis (assuming Cryothecomonas is a junior synonym of Protaspis)? Currently, we do not think that we can generalize our ultrastructural results for P. grandis to be characteristic for all Protaspis species because there are no further TEM observations for the genus. It is highly likely that all species have a nucleus with permanently condensed chromosomes, because the granular appearance of the nucleus is readily visible with light microscopy (which gave rise to previous classifications of Protaspis within the dinoflagellates and euglenids, see the Introduction). The possession of a multilayered wall and flagellar insertion funnels should be verified by ultrastructural investigations of the other Protaspis species. As circumscribed today, Protaspis seems to be a welldefined genus. However, this is not the case for Cryothecomonas, and we argue that it is an artificial (polyphyletic) genus for the following reasons. The type of flagellar insertion, the flagella motion and the site of pseudopodia formation are different within the genus (Drebes et al. 1996; Schnepf and Ku¨hn 2000; Thomsen et al. 1991). The type species Cryothecomonas armigera Thomsen et al. 1991 has apically inserting isodynamic flagella separated by a papilla (Thomsen et al. 1991; Table 1 and Fig. 34). The pseudopodia emerge from a slit (cytostome) located posterio-laterally (Thomsen et al. 1991; Table 1 and Fig. 34). All other species in the genus that were described together with the type species, namely Cryothecomonas inermis, Cryothecomonas scybalophora,

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and Cryothecomonas vesiculata, share these features (Thomsen et al. 1991; Table 1). Cryothecomonas aestivalis Drebes, Ku¨hn, and Schnepf, 1996 has apically inserting heterodynamic flagella separated by a papilla, and the pseudopodia emerge from the posterior cell pole (Drebes et al. 1996). Cryothecomonas longipes has the anterior flagellum inserting apically, the posterior flagellum inserting subapically (heterodynamic), and a rarely noticeable papilla (Schnepf and Ku¨hn 2000; Table 1 and Fig. 34). The pseudopodia emerge from a ventral slit in the anterior cell half (Schnepf and Ku¨hn 2000; Table 1). If we stress the importance of differences in the flagellar insertion organization and patterns of motility and view them as diagnostic characters at the generic level, then the genus Cryothecomonas should be split into three genera, with C. aestivalis and C. longipes each representing a new genus. However, we hesitate to formally split the genus at this early stage, because there are no sequence data yet available for any Cryothecomonas species that shares the same characters as the type. The SSU rDNA sequence for the type species, C. armigera, should be added to the existing dataset before nomenclatural changes are recommended. Nonetheless, if the current members of Cryothecomonas were split into three genera, then they would all be distinguishable from Protaspis on the basis of flagellar insertion organization, with the new ‘‘C. longipes-genus’’ being most closely related to it. Alternatively, it might be more appropriate to de-emphasize slight modifications of the configuration of flagellar insertions (e.g. C. longipes versus P. grandis) and transfer all species of Cryothecomonas into Protaspis, making the former a junior synonym of the latter. This would be the best approach to take if at some point it is demonstrated that all of these species are intermingled within a robustly supported clade inferred from SSU rDNA sequences or a comparable molecular marker. Although there are no data for the type species of Protaspis yet, it is likely that all of these species whether placed in one or up to four different genera will share the following features: (1) mitochondria with tubular cristae; (2) nucleus with permanently condensed chromosomes and conspicuous nucleolus; (3) multilayered cell wall outside of the plasma membrane; (4) feeding by means of pseudopodia; and (5) flagella emerging through funnels. Molecular phylogeny and further expansion of the Cercozoa. Our phylogenetic analyses demonstrated that P. grandis is closely related to C. longipes. The sequence from P. grandis represents only the third reference taxon (morphologically described species) within the Cryomonadida clade, which is currently comprised mainly of unidentified environmental sequences (Fig. 33). A phylogenetic analysis excluding the shorter environmental sequences and including only morphologically described taxa within the Cercozoa (including P. grandis) is presented elsewhere (Hoppenrath and Leander 2006). Previous SSU rRNA phylogenies have shown that Cryothecomonas clusters robustly within the Cercozoa and have helped clarify the relative relationships between Cryothecomonas, Heteromita, 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).

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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 sampling of heterotrophic amoeboflagellates that potentially belong to the Cercozoa make it difficult to infer correct relationships

among them. Gaining new sequences from microscopically identified and characterized species (e.g. P. grandis) should provide new insights into the evolutionary history and diversity of

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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.

32.5–55 20–35 No No Posterior-central

08–32 10–27 Yes-left to median Yes Anterior median

Spherical No Unreported Yes Posterior half, median No Unreported Unreported Unreported Gliding Yes (lmc only) Unreported Benthic

Nucleus shape Nuclear cap Condensed chromosomes Nucleolus Ventral furrow/groove Pseudopodia Feeding mode Body scales Flagella scales Movement Cell wall Extrusomes Habitat

Spherical No Yes Yes Anterior half, median Yes Phagotrophy No No Gliding Yes, multilayered Yes Benthic

 0.5 cl  cl Oval-rectangular Smooth Dorso-ventral

Subapical, funnel Protrusion

P. grandis n. sp.

 0.5–0.75 cl  0.5–1.5 cl Round-oval Smooth Dorso-ventral

Subapical Protrusion

P. obliqua

Flagellar length Anteriora Posteriora Cell shape Cell surface Cell flattening Cell size (mm) Length Width Anterior indentation Posterior indentation Nucleus position

Flagella insertion

Characters

Spherical No Unreported Unreported  Full cell length, median Yes Phagotrophy Unreported Unreported Gliding Unreported Unreported Benthic, planktic

12–30 09–15 No No Anterior or central lateral

 0.5–1  cl 1.2–2  cl Oval Smooth Dorso-ventral

Subapical No

P. glans

Spherical No/yesb Unreported Unreported Indistinct Yes Phagotrophy Unreported Unreported Gliding Unreported Unreported Benthic

08–17 08–12 No No Anterior median

 cl  1.3–3  cl Round-oval Warty Dorso-ventral

Subapical No

P. gemmifera

Table 1. Comparison of morphological character states in described species of Protaspis and Cryothecomonas.

14–25 08–14 No No Anterior, median or right-hand side Spherical No/yesb Unreported Unreported  Full cell length, median Yes Phagotrophy Unreported Unreported Gliding Unreported Unreported Benthic, planktic

 1–2  cl  1.5–2.5  cl Oblong-ovate Smooth or warty Dorso-ventral

Subapical No

P. tegere

Discoidal No/yesb Unreported Unreported  Full cell length, median Unreported Unreported Unreported Unreported Gliding Unreported Unreported Benthic, planktic

09–22 8.5–16 No No Anterior median

 cl  1.3–2  cl Round-oval Fine warts Dorso-ventral

Subapical No

P. verrucosa

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Table 1. (Continued).

Nucleus shape Nuclear cap Condensed chromosomes Nucleolus Ventral furrow/groove Pseudopodia Feeding mode Body scales Flagella scales Movement Cell wall Extrusomes Habitat

Flagellar length Anteriora Posteriora Cell shape Cell surface Cell flattening Cell size (mm) Length Width Anterior indentation Posterior indentation Nucleus position

Flagella insertion

Characters

P. maior

P. metarhiza

 0.5–0.3 cl  cl Oval, posterior tip Smooth Dorso-ventral 28–38 16–27 No No Right lateral, Anterior–central Spherical-ovoid No Unreported Unreported  cell length, left from median Yes Phagotrophy Unreported Unreported Gliding, swim Unreported Unreported Planktic

24–40 16–30 No No Anterior median, Antero-lateral Spherical No Unreported Yes  cell length, oblique Yes Phagotrophy Unreported Unreported Gliding Unreported Unreported Benthic, planktic

Subapical No

 cl 2  cl Oval Smooth Dorso-ventral

Subapical No

P. obovata

Spherical No Yes Unreported  cell length, straight or sigmoid Yes Phagotrophy Unreported Unreported Swim, rotational Unreported Unreported Planktic

26–40 17–25 No, papilla No, truncate Anterior

 cl  cl Ovoid (pear) Smooth Dorso-ventral

Subapical No

P. simplex

4.5–25 02–10 No No Anterior, Antero-lateral Ovoid Yes, in some cells Unreported Yes Shallow, median Not observed Unreported Unreported Unreported Gliding, wobbling Unreported Unreported Benthic, planktic

 0.5–1 cl 1–3  cl Round-oval-ovoid Fine warts in some Dorso-ventral

Subapical No

P. tanyopsis

Unreported Unreported Planktic

Spherical No Unreported Unreported  2/3 Cell length Not observed Unreported Unreported Unreported

28–30 09–11 No No Anterior

 1.2 cl  1.3 cl Elongate Unreported Dorso-ventral

Subapical Protrusion

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Table 1. (Continued).

Cell shape Cell surface Cell flattening Cell size (mm): Length Width Nucleus position Nucleus shape Nuclear cap Condensed chromatin Nucleolus Furrow/groove Cytostome/slit Pseudopodia Feeding mode Body scales Flagella scales Movement Cell wall/theca Extrusomes Muciferous bodies Habitat

Flagellar length Anterior Posterior

Flagellar insertion

Characters

09–15 mm 20–24 mm, fine hairs 25 mm Heterodynamic Oval, kidney-shaped Smooth Slightly 09–14 07–09 Anterior Round, lobed Unreported Yes Yes Slit, ventral left side, 2/3 cell length Yes, anterior Yes Phagotrophy No No Swim Yes, multilayered Yes No Planktic

Apical & subapical, Funnels, papillad

C. longipes

15 mm 25 mm Heterodynamic Oblong-oval Smooth No 09–12 04–05 Anterior Oval, lobed Unreported Yes Yes Unreported 0.5–1 cell length Yes Yes, posterior Phagotrophy No No Swim, glide Yes, bilayered No No Planktic

Apical, funnels, Papilla

C. aestivalis

1–2  cell length 1–2  cell length Isodynamic Egg-shaped Smooth Yes 12–32 07–23 Anterior Round-oval Unreported Yes Yes Lateral, 0.5–1cell length Yes, posterio-lateral Yes Phagotrophy No No Unreported Yes, multilayered Yes Yes Planktic, sea ice

Apical, funnels, Papilla

C. armigera

1–2  cell length Unreported Isodynamic Egg-shaped Smooth Slightly 10–15 07–10 Anterior Round-oval Unreported Yes Yes Lateral Yes, posterio-lateral Yes Phagotrophy No No Unreported Yes, multilayered No Yes Planktic, sea ice

Apical, funnels, Papilla

C. inermis

Unreported Unreported Unreported Highly variable With debris No 09–14 4.5–09 Anterior Round Unreported Yes Yes Lateral Yes, posterio-lateral Yes Phagotrophy No No Unreported Yes, monolayered Yes Yes Planktic, sea ice

Apical, funnels, Papilla

C. scybalophora

Unreported Unreported Unreported Elongated Protuberances Slightly 10–15 4–08 Anterior Round-oval Unreported Yes Yes Lateral Yes Yes Phagotrophy No No Unreported Yes, bilayered Probably Yes, many Planktic, sea ice

Apical, funnels, Papilla

C. vesiculata

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cercozoans (Ekelund, Daugbjerg, and Fredslund 2004; Ku¨hn, Medlin, and Eller 2004). The Cercozoa was erected on the basis of molecular phylogenetic data alone (Cavalier-Smith 1998a, b); no morphological feature characterizes the whole group and the taxonomic diagnosis is unusually broad (Cavalier-Smith 1998a, p. 232). During the last 7 years, the identity and composition of the Cercozoa has been continuously modified (Bass and Cavalier-Smith 2004; Cavalier-Smith and Chao 2003; Hoppenrath and Leander 2006). The ‘‘undescribed cercozoan clade II’’ in Fig. 33 is the sister clade to the Cryomonadida and was shown to comprise ebriids (Hoppenrath and Leander 2006). This resulted in the necessity to amend the diagnosis for the Cercozoa to include taxa with internal siliceous skeletons. Our observations of P. grandis require additional amendments to the diagnosis of the clade in order to accommodate taxa with a rigid protein layer outside of the plasma membrane (e.g. the cryomonads Protaspis and Cryothecomonas). These changes make the diagnosis for the clade even broader and make the prospect of defining the clade on morphological grounds alone even more remote.

ACKNOWLEDGMENTS We wish to thank W. J. Lee and D. J. Patterson for discussions on a part of the manuscript and help with the literature. We would like to acknowledge discussions with Ø. Moestrup about the taxonomic importance of flagellar insertions patterns and thank G. Eller who generously provided a provisional alignment of small subunit rDNA sequences (‘‘Pseudopirsonia-alignment’’). This work was supported by a scholarship to M. Hoppenrath from the Deutsche Forschungsgemeinschaft (grant Ho3267/1-1) and by grants to B. S. Leander from the National Science and Engineering Research Council of Canada (NSERC 283091-04) and the Canadian Institute for Advanced Research. B. S. Leander is a Scholar of the Canadian Institute for Advanced Research, Program in Evolutionary Biology.

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Received: 12/04/05, 03/30/06, 04/03/06; accepted: 04/03/06

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