J. Eukaryot. Microbiol., 53(5), 2006 pp. 327–342 2006 … · Molecular Phylogenetic Position of Protaspis grandis n. sp. MONA HOPPENRATH and BRIAN S. LEANDER ... Patterson and Zo¨lffel

Dinoflagellate, Euglenid, or Cercomonad? The Ultrastructure andMolecular 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 severaldifferent groups of eukaryotes, including dinoflagellates, euglenids, and cercomonads. This uncertainty led us to investigate thephylogenetic position of Protaspis grandis n. sp. with ultrastructural and small subunit (SSU) rDNA sequence data. Our resultsdemonstrated that the cells were dorsoventrally flattened, shaped like elongated ovals with parallel lateral sides, 32.5–55.0mm long and20.0–35.0mm wide. Moreover, two heterodynamic flagella emerged through funnels that were positioned subapically, each within adepression 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 containingnumerous mitochondria with tubular cristae emerged from a ventral furrow through a longitudinal slit that was positioned posterior to theprotrusion and flagellar apparatus. Batteries of extrusomes were present within the cytoplasm and had ejection sites through pores in thecell wall. The SSU rDNA phylogeny demonstrated a very close relationship between the benthic P. grandis n. sp. and the planktonicCryothecomonas longipes. These ultrastructural and molecular phylogenetic data for Protaspis indicated that the current taxonomy ofProtaspis and Crythecomonas is in need of re-evaluation. The composition and identity of Protaspis is reviewed and suggestions for futuretaxonomic changes are presented. Problems within the genus Cryothecomonas are highlighted as well, and the missing data needed toresolve ambiguities between the two genera are clarified.

Key Words. Cercozoa, Cryothecomonas, morphology, phylogenetic analysis, Protaspis, SSU rDNA, taxonomy, ultrastructure.

THE genus Protaspis was described by Skuja (1939), with thetype species Protaspis glans and two additional species,

Protaspis maior and Protaspis metarhiza (Skuja 1939). Skuja(1939) erected the new family Protaspidaceae for his new genusand classified it tentatively within the euglenids, because of thestrong continuous ‘‘periplast,’’ the ventral longitudinal furrow,the two heterodynamic flagella, the large nucleus with nucleolusand the paramylon-like reserve product. The flagella insert in theanterior part of the ventral furrow and are clearly separated fromeach other. The movement is by gliding and food uptake is bypseudopods formed out of the furrow (Skuja 1939). Skuja (1948)described a fourth species, Protaspis obovata. The large nucleusof this species had the characteristic morphology of a dinoflagel-late nucleus (dinokaryon), which led Skuja (1948) to reclassifyProtaspis within the Pyrrophyta (dinoflagellates). This unusualcombination of characters led Skuja (1948) to entertain thepossibility that protaspids might occupy an intermediate phylo-genetic position between euglenids and dinoflagellates.

From the time of Skuja’s work to the early 1990s, Protaspiswas consistently treated as a dinoflagellate taxon (Chretiennot-Dinet 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 andstated that it is a problematic genus, possibly of euglenid affinity.However, at about that same time, Protaspis was also tentativelyclassified as belonging to the Thaumatomastigaceae/Thaumato-mastigidae (Larsen and Patterson 1990; Patterson et al. 2002;Patterson and Zolffel 1991). Mylnikov and Karpov (2004) arguedthat Protaspis should be transferred into the Cercomonadidabecause protaspids do not have a flagellar pocket and unlikethaumatomonads, do not have body scales. This opinion isreflected in the latest higher-level classification of eukaryotes,where Protaspis is classified in the Cercomonadida in the‘‘Family’’ Heteromitidae (Adl et al. 2005).

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 V�rs 1995/96; Larsen 1985; Larsen and Patterson 1990; Lee and Patterson2000; Lee et al. 2003, 2005; Norris 1961; Patterson et al. 1993;Skuja 1939, 1948; Tong et al. 1998; V�rs 1992, 1993). Currently,the genus contains 10 species: P. glans Skuja 1939, P. maiorSkuja 1939, P. metarhiza Skuja 1939, P. obovata Skuja 1948,Protaspis tanyopsis Norris 1961, Protaspis gemmifera Larsen andPatterson 1990, Protaspis obliqua Larsen and Patterson 1990,Protaspis tegere Larsen and Patterson 1990, Protaspis verrucosaLarsen and Patterson 1990, and Protaspis simplex V�rs 1992. Thedistinguishing features among some of these species are not clear,and it is likely that some Protaspis species will prove to beconspecific (for detailed discussion in Lee 2001). The unresolvedphylogenetic position of Protaspis and the potential affiliation todinoflagellates or euglenids motivated us to investigate the phy-logeny of a new species, Protaspis grandis n. sp., on the basis ofultrastructural and small subunit (SSU) rDNA sequence data.

MATERIALS AND METHODS

Collection of organisms. Samples were collected with aspoon during low tide at Centennial Beach, Boundary Bay, BC,Canada. The salinity of the water is about 30–33 psu. The sandsamples were transported directly to the laboratory and theflagellates were separated from the sand by extraction through a45-mm filter using melting seawater-ice (Uhlig 1964). The flagel-lates accumulated in a Petri dish beneath the filter and were thenidentified with an inverted-microscope at 40 � –250 � magnifi-cation. Cells were isolated by micropipetting and used directly(not from culture) for the preparations described below.

Light microscopy. Cells were observed directly and micro-manipulated with a Leica DMIL inverted microscope. For differ-ential interference contrast light microscopy, micropipetted cellswere placed on a glass specimen slide and covered with a coverslip. Images were produced with a Zeiss Axioplan 2 imagingmicroscope connected to a Leica DC500 color digital camera.

Scanning electron microscopy. A mixed-extraction samplewas fixed overnight with two drops of acidic Lugol’s solution.Cells were transferred onto a 5-mm polycarbonate membrane filter

Corresponding Author: M. Hoppenrath, Canadian Institute for Ad-vanced Research, Program in Evolutionary Biology, Departments ofBotany 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]

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J. Eukaryot. Microbiol., 53(5), 2006 pp. 327–342r 2006 The Author(s)Journal compilation r 2006 by the International Society of ProtistologistsDOI: 10.1111/j.1550-7408.2006.00110.x

(Corning Separations Div., Acton, MA), washed with distilledwater, dehydrated with a graded series of ethanol and critical pointdried with CO2. Filters were mounted on stubs, sputter-coatedwith gold and viewed under a Hitachi S4700 Scanning ElectronMicroscope (SEM). Some SEM images were presented on a blackbackground using Adobe Photoshop 6.0 (Adobe Systems, SanJose, CA).

Transmission electron microscopy. Cells were concentratedin a microfuge tube by micropipetting and slow centrifugation.The pellet of cells was prefixed with 2% (v/v) glutaraldehyde inseawater at 4 1C for 30 min. Cells were washed twice in filteredseawater (30–35 psu) before post-fixation in 1% (w/v) OsO4 inseawater for 30 min at room temperature. Cells were dehydratedthrough a graded series of ethanol, infiltrated with acetone–resinmixtures (pure acetone, 2:1, 1:1, 1:2, pure resin), and embedded inpure resin (Epon 812). The block was polymerized at 60 1C andsectioned with a diamond knife on a Leica Ultracut UltraMicro-tome. Thin sections were post-stained with uranyl acetate and leadcitrate and viewed under a Hitachi H7600 Transmission ElectronMicroscope.

DNA extraction, amplification, cloning and squencing. Fortyindividually isolated (uncultured) cells were washed three times infiltered (eukaryote-free) seawater. Genomic DNA was extractedfrom the 40-cell preparation using a standard hexadecyltrimethy-lammonium bromide (CTAB) extraction protocol (Zolan andPukkila 1986). The PCR amplification protocol using universaleukaryotic primers reported previously (Leander, Clopton, andKeeling 2003) consisted of an initial denaturing period (95 1Cfor 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 finalextension period (72 1C for 5 min). Polymerase chain reactionproducts corresponding to the expected size were gel isolatedand cloned into the pCR2.1 using the TOPO TA cloning kit(Invitrogen, Burlington, ON, Canada). Two clones were sequencedwith ABI big-dye reaction mix using the vector primers andinternal primers oriented in both directions.

Phylogenetic analyses. The Protaspis SSU rDNA sequencewas aligned with other eukaryotic sequences using MacClade 4(Maddison and Maddison 2000) forming a 54-taxon alignment.Maximum likelihood (ML), ML distance, and Bayesian methodsunder different DNA substitution models were performed. Allgaps were excluded from the alignments before phylogeneticanalysis. The a shape parameters were estimated from the datausing HKY under a g distribution plus invariable sites model andfour rate categories (a5 0.28, Ti/Tv 5 1.38, fraction of invariablesites 5 0.08). A gamma-corrected ML tree (analyzed using theparameters listed above) was constructed with PAUP� 4.0 usingthe general time reversible (GTR) model for base substitutions(Posada and Crandall 1998; Swofford 1999). Gamma correctedML tree topologies found with HKY and GTR were identical.Maximum likelihood bootstrap analyses were performed inPAUP� 4.0 (Swofford 1999) on 100 re-sampled datasets underan HKY model using the a shape parameter, proportion ofinvariable sites and transition/transversion ratio (Ti/Tv) estimatedfrom the original dataset.

Maximum likelihood distances for the SSU rDNA datasetwere calculated with TREE-PUZZLE 5.0 using the GTR sub-stitution matrix (Strimmer and Von Haeseler 1996). A distancetree was constructed with weighted neighbor joining (WNJ)using Weighbor (Bruno, Socci, and Halpern 2000). Five-hundredbootstrap datasets were generated with SEQBOOT (Felsenstein1993). Respective distances were calculated with the shellscript ‘‘puzzleboot’’ (M. Holder and A. Roger, www.tree-puz-zle.de) using the a shape parameter and proportion of invariablesites estimated from the original dataset and analyzed withWeighbor.

We also examined the dataset with Bayesian analysis using theprogram MrBayes 3.0 (Huelsenbeck and Ronquist 2001). Theprogram was set to operate with GTR, a g distribution and fourMCMC chains starting from a random tree (default tempera-ture 5 0.2). A total of 2,000,000 generations were calculated withtrees sampled every 100 generations and with a prior burn-in of200,000 generations (2,000 sampled trees were discarded). Amajority rule consensus tree was constructed from 16,000 post-burn-in trees with PAUP� 4.0. Posterior probabilities correspondto the frequency at which a given node is found in the post-burn-intrees.

GenBank accession numbers. Allas diplophysa (AF411262),Allas sp. (AF411263), Bigelowiella natans (AF054832), Cerco-monas longicauda (AF101052), Cercomonas sp. (U42448), Cer-cozoa sp. WHO1 (AF411273), Chlorarachnion reptans (U03477),Cryothecomonas aestivalis (AF290541), Cryothecomonas long-ipes (AF290540), Euglypha rotunda (AJ418784), Gromia ovifor-mis (AJ457813), Heteromita globosa (U42447), Lotharellaglobosa (AF076169), Massisteria marina (AF174372), Paulinellachromatophora (X81811), Phagomyxa bellerochea (AF310903),Polymyxa betae (AF310902), P. grandis n. sp. (DQ303924),Pseudodifflugia cf. gracilis (AJ418794), Pseudopirsonia mucosa(AJ561116), Thaumatomastix sp. (AF411261), Thaumatomonasseravini (AF411259), Thaumatomonas sp. (U42446), Thaumato-monas sp. (SA) (AF411260), uncultured cercozoan (AF372764),uncultured cercozoan (AY180012), uncultured cercozoan(AY180035), uncultured cercozoan (AY620274), uncultured cer-cozoan (AY620276), uncultured cercozoan (AY620277), uncul-tured cercozoan (AY620279–AY620281), uncultured cercozoan(AY620294), uncultured cercozoan (AY620295), uncultured cer-cozoan (AY620300), uncultured cercozoan (AY620314), uncul-tured cercozoan (AY620316), uncultured cercozoan (AY620320–AY620323), uncultured cercozoan (AY620340), unculturedcercozoan (AY620348–AY620353), uncultured cercozoan(AY620355), uncultured cercozoan (AY620357), unculturedeukaryote TAGIRI-2 (AB191410), uncultured eukaryote(AJ130856), uncultured eukaryote (AY082998).

RESULTS

Cell shape and flagella. The flagellated stage in the life cycleof P. grandis was investigated. Plasmodial and cyst stages werenot 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 ovalswith parallel lateral sides (Fig. 1–4). The ventral side of the cellwas concave and the dorsal side was slightly convex (Fig. 5). Twothin, heterodynamic flagella emerged from separate subapicaldepressions on the ventral side of the cell (Fig. 4, 11). The anteriorflagellum was about half a cell length, and the trailing flagellumwas about one cell length. A protrusion emerging from the rightfurrow border pointed to the left and separated the insertiondepressions of the flagella (Fig. 1, 4, 11, 15). The posteriorflagellum inserted below the protrusion and was positionedslightly to the left. A narrow ventral furrow is positioned mediallyand extends about one-third of the cell length posteriorly from theinsertion point of the posterior flagellum and towards the posteriorend (Fig. 1, 3, 4, 11). The flagella were anchored within flagellarpits and emerged through funnels with a sharp outer margin andring-shaped basal depression (Fig. 16). Transmission electronmicrographs through this region of the cell contained a structurethat lacked the typical 912 organization of microtubules and wascomposed of material arranged in concentric rings of differentdensity (Fig. 17). This morphology is consistent with both aflagellum, in transverse view, as seen in C. aestivalis (Drebeset al. 1996, Fig. 23) and an extrusome, in transverse view.

328 J. EUKARYOT. MICROBIOL., VOL. 53, NO. 5, SEPTEMBER– OCTOBER 2006

Nucleus and cytoplasm. A large, round nucleus with a gran-ular appearance was situated in the posterior half of the cell andmidway between the lateral margins (Fig. 2, 4, 5). The nucleuswas never observed in a lateral position or in the anterior half ofthe cell. Nuclear caps were not observed. The nucleus contained alarge nucleolus and permanently condensed chromosomes dis-tributed evenly (Fig. 12, 13). Some of our micrographs showed aconvoluted cleft in the anterior side of the nucleus (Fig. 13). Thecytoplasm was generally colorless and contained food vacuoles,masses of small vesicles/granules, and often some larger coloredparticles (e.g. yellow, orange, red, or brown) (Fig. 1–4, 12, 13).The ultrastructure of the large colored particles was uniform andoften darkly stained suggesting that they were globules of lipids(Fig. 12, 13). Mitochondria were evenly distributed throughoutthe cytoplasm and had tubular-shaped cristae (Fig. 14). Thecytoplasm also contained elongated Golgi bodies composed ofstacks of about six to nine cisternae (Fig. 20).

Ventral furrow and pseudopodia. In some cells, relativelylong and thin pseudopodia emerged from the ventral furrow andspread over the substrate surface (not shown). An unidentifiedProtaspis cell with typical extruded pseudopodia is shown in Fig.10. Pseudopodia were evident in our P. grandis samples preparedfor SEM and TEM as spherical globules located near the ventralfurrow (Fig. 18, 19). The ventral furrow was observed in threedifferent 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). Thepseudopodia were bordered by the plasma membrane, lacked acell wall, and were rich in mitochondria (Fig. 25). Before cell

division, the flagellar apparatus doubled (Fig. 26). Although celldivision was not observed during this study, the pattern of flagellarreplication indicated that longitudinal division most likely occursalong a longitudinal cleavage furrow, which has also been de-scribed for other species in the genus.

Cell wall. The cell surface was smooth and reinforced with athickened cell wall comprised of at least seven different layers ofmaterial having different thicknesses, densities, and architectures(Fig. 2, 3, 11, 12, 15, 27, 28). The multilayered wall was secretedoutside of the plasma membrane (Fig. 28). An amorphous ‘‘basallayer’’ about 150 nm thick was positioned directly above theplasma membrane and was overlain by a broad ‘‘intermediatelayer’’ about 400 nm thick (Fig. 28). A ‘‘vesicular layer’’ (50 nmthick) interrupted the relatively homogeneous intermediate layernear its superficial margin (Fig. 28). A thin electron-dense ‘‘outerlamina’’ was squeezed between the intermediate layer and asealing ‘‘coat’’ consisting of three ridged layers: a darker ‘‘deepcoat,’’ a columnar ‘‘mid-coat,’’ and a parallel ‘‘superficial coat’’(Fig. 27, 28). In tangential section, the regular spacing of theridges of the coat were easily discernable (Fig. 29). Cylindricalpores pierced the basal and intermediate layers and permittedextrusomes 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 wereusually concentrated in clusters (Fig. 31), and each extrusome waslocated within a membrane-bound vesicle (Fig. 32).

Molecular phylogeny. We sequenced the SSU rRNA genefrom a multi-specimen sample of P. grandis collected in Septem-ber 2004. Basic local alignment search tool results robustly placed

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 elongatedoval cell shape, granular cytoplasm, ventral furrow near the anterior end (arrowhead), and an anterior protrusion (arrow) stemming from the right marginof 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 closedventral furrow (arrowhead). 4. Ventral view of P. grandis n. sp. showing the nucleus (n), an open ventral furrow, and the insertion point of the posteriorflagellum (arrowhead) within a depression that is positioned below the anterior protrusion (arrow). 5. Lateral view of P. grandis n. sp. showing theconcave 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, medianfocus, 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. obliquashowing the posterior indentation (arrowhead). 9. Ventral view of P. obliqua showing the anterior indentation (arrow). 10. An unidentified Protaspisspecies showing the typical morphology and distribution of pseudopods over the substrate. (Fig. 1–5, Bar 5 10 mm; Fig. 6–9, Bar 5 10mm; Fig. 10,Bar 5 10mm).

329HOPPENRATH & LEANDER—MORPHOLOGY AND PHYLOGENY OF PROTASPIS GRANDIS


the sequence together with C. longipes and several related envir-onmental sequences, all of which belonged to the ‘‘Cercozoa.’’We created a 54-taxon alignment (1,006 unambiguous sites)consisting of ingroup cercozoans and representative sequencesfrom plasmodiophorids, chlorarachniophytes, and Gromia (Fig.33). Protaspis grandis, C. longipes, and three environmentalsequences (AY620323, AY620340, and AY620352) branchedtogether with strong support from both bootstrap statistics andBayesian posterior probabilities (Fig. 33). This subclade fellwithin a much larger clade containing C. aestivalis and severalenvironmental sequences, all of which formed the well-supportedCryomonadida clade. Interestingly, C. longipes branched muchmore closely to P. grandis than to C. aestivalis (Fig. 33). None-theless, the Cryomonadida clade formed the sister group toa sequence clade of unknown identity, namely ‘‘undescribedcecozoan clade II’’ and was only distantly related to the Thau-matomonadida (Fig. 33).

DISCUSSION

Comparative morphology. The overall light-microscopicalappearance of the species described here conforms with thecharacteristic features for the genus Protaspis: heterodynamicflagella insert subapically within separate depressions on theventral surface of the cell, movement is by substrate-mediatedgliding, feeding is by means of pseudopodia that emerge from aventral furrow and the granularity of the nucleus is clearly visible.Protaspis grandis n. sp. is larger than all other described species inthe genus, but its smallest size overlaps with the largest size rangeof the larger species, P. maior, P. metarhiza, and P. obovata(Table 1). Protaspis grandis n. sp. is clearly distinguishable fromthese species and nearly all other Protaspis species by having ananterior protrusion that separates the flagellar insertion points andan obviously thick cell wall (Fig. 34). Only P. tanyopsis and P.obliqua have a similar protrusion (Larsen and Patterson 1990; Leeand Patterson 2000; Lee, Simpson, and Patterson 2005; Norris1961). Protaspis tanyopsis also has a similar shape to P. grandisbut is smaller, only slightly flattened dorso-ventrally, and lacks athickened cell wall. Moreover, P. tanyopsis is longer than P.grandis relative to the cell width and the nucleus is positionedanteriorly rather than posteriorly (Norris 1961; Table 1 and Fig.34). Norris (1961) described P. tanyopsis as having a small lobenear the anterior end of the groove margin but did not specifywhich margin, the left or right side of the groove. Although weremain doubtful, his drawing suggests that the lobe/protrusion ispart of the left-hand groove margin (Fig. 34). By contrast, theanterior protrusion of P. grandis and P. obliqua emerges from theright-hand furrow margin. Like P. grandis, P. obliqua also has athickened cell wall (Larsen and Patterson 1990; Lee 2001; Leeand Patterson 2000; Lee et al. 2005). However, the cells of P.obliqua are significantly smaller than P. grandis, are round to ovalrather than elongated, and have a posterior indentation that gives

the cell an asymmetrical appearance (Fig. 34). Moreover, thefurrow of P. obliqua is in the posterior half of the cell andthe nucleus is positioned in the anterior half, which stands incontrast to the anterior furrow and posterior nucleus positionin P. grandis (Larsen and Patterson 1990, 2000; Table 1;Lee 2001 and Fig. 34). These morphological characters clearlydistinguish P. obliqua from P. grandis, and we were always ableto distinguish these two species in samples in which both of thesespecies co-occurred (Fig. 1–9). The P. obliqua cells were 27.5–35.0mm long and 25.0–32.5 mm wide (n 5 10), which is slightlylarger than that reported in the literature (Table 1). No intermedi-ate morphologies between P. obliqua and P. grandis were ob-served in our samples. Therefore, we establish Protaspis grandisn. sp. and provide the following species description.

Taxon Description

Cercozoa Cavalier-Smith 1998, emend. Adl et al. 2005Cercomonadida Poche 1913, emend. Vickerman 1983, emend.

Mylnikov 1986Heteromitidae Kent 1880, emend. Mylnikov 1990, emend.

Mylnikov and Karpov 2004Protaspis Skuja 1939

Protaspis grandis Hoppenrath et Leander n. sp.

Diagnosis. Cells shaped as elongated ovals with parallel lateralsides, dorsoventrally flattened, 32.5–55.0 mm long, 20.0–35.0mmwide. Two heterodynamic flagella, inserted subapically, separatedby a protrusion. Flagellar pits with funnel. Ventral longitudinalfurrow in the anterior half of cell. Nucleus in the posterior half ofcell, with nucleolus and permanently condensed chromosomes.Pseudopodia emerge through a slit within the ventral furrow.Thickened cell wall outside of the plasma membrane consistingof seven layers: basal layer, intermediate layer, vesicular layer,outer lamina, deep coat, mid-coat, and superficial coat. Basal layerand intermediate layer pierced by pores for the discharge ofextrusomes.

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, andAugust 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 differentspecies? V�rs (1992) stated that the species are ‘‘. . . distinguishedby size and shape, the length and path of the groove, and thepresence or absence of (1) a nuclear cap, (2) a protrusion separat-ing 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 (left-hand cell) and a ventral surface view (right-hand cell) containing the anterior protrusion (double arrowhead) and longitudinal ventral slit (arrow); theventral 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, largelipid globules (lg) posterior to the nucleus, and vesicular cytoplasm (v) anterior to the nucleus (Bar 5 2 mm). 13. TEM showing a nucleus with aconvoluted cleft (arrow), a food vacuole (fv), and darkly stained lipid globules (l) (Bar 5 2mm). 14. TEM showing a typical double membrane-boundmitochondrion with tubular cristae (Bar 5 0.25 mm). 15. SEM showing the anterior protrusion (double arrowhead) separating the depressions withinwhich the heterodynamic flagella originate (arrows). The shortness of flagella is a common artifact in SEM preparation. The ventral slit (arrowhead) isevident within the ventral furrow (Bar 5 2mm). 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 inconcentric rings. This morphology is consistent with both a flagellum, in transverse view, as seen in Cryothecomonas aestivalis and an extrusome, intransverse view (Bar 5 0.1 mm).



and a warty cell surface’’ (p. 91). It has been noticed that the sizerange of Protaspis species was often expanded when furtherobservations of a species were made in different habitats (e.g.Lee 2001; Lee and Patterson 2000). Most of the Protaspis speciesshow considerable overlap in their size ranges (Table 1), andtherefore cell size is only of value when the differences arerelatively discrete and are used in connection with other features.The flattened cell shape of most of Protaspis species is verysimilar (round to oval to ovoid; Table 1 and Fig. 34), and mostlikely there is a limited degree of variation (Table 1) dependingon the age of the cell, feeding stages and cell cycle stages(e.g. P. obovata in Skuja 1948). The cell shape must differsignificantly 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 usefulcharacter, 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 thecell length, the width (e.g. broad versus widening at the endsversus narrow), and the position (e.g. indistinct versus whole celllength versus mainly anterior versus mainly posterior) of thefurrow appear to be reasonable diagnostic characters (Table 1).The presence or absence of nuclear caps is not a consistent featureor has not been recorded reliably (Tong et al. 1998); P. gemmifera,P. tegere, and P. verrucosa were all described both with andwithout them (Larsen and Patterson 1990; Lee 2001; Lee andPatterson 2000; Patterson et al. 1993; Tong et al. 1998). Theanterior protrusion separating the flagella is still regarded as aconsistent species character. Special cell inclusions like conspic-uous reserve material (e.g. rod-shaped in P. gemmifera) are nolonger used as a characteristic feature (Lee 2001; Lee andPatterson 2000). The presence or absence of warts on the cellsurface 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; Leeand Patterson 2000; Lee et al. 2005; V�rs 1992). The position ofthe nucleus is nearly the same in all Protaspis species (Table 1 andFig. 34) and varies to a certain extent in P. metarhiza and P. glansdepending on the age of the cells (Larsen 1985; Larsen andPatterson 1990; Skuja 1939). Protaspis grandis is the only speciesdescribed so far with the nucleus positioned in the posterior half ofthe cell. The thickness of the cell wall is a distinguishing feature,with only P. obliqua and P. grandis having a thick one that iseasily visible with light microscopy (Larsen and Patterson 1990;Lee and Patterson 2000; Fig. 1–9).

A posterior (and anterior?) indentation seems to be a specialfeature, so far only described for P. obliqua (Larsen and Patterson1990; Lee 2001; Lee and Patterson 2000). Moreover, there is largeand overlapping range for flagellar lengths within a species (Table1), making it useless for species identification. The mode offlagellar movement could be of interest, but this behavioralcharacter has not been sufficiently documented for all of thedescribed species. The possibility of different cell behaviorsduring different feeding and cell cycle stages would make theuse of these characteristics for species separation challenging.

Pseudopodia are also difficult to observe and the morphologicaldetails associated with these structures are not available for all thespecies, which questions the taxonomic value of these features.Therefore, in our view, characters like anterior protrusions, ante-rior or posterior indentations, furrow morphology, and the relativethickness of the cell wall are the best diagnostic features currentlyavailable. In addition, the position of the nucleus within the cell(e.g. anterior versus posterior) and cell shape (round versuselongated) used in connection with discrete differences in therange of cell sizes appear to be practical ‘‘combination-characters’’ (Fig. 34).

After taking the above discussion about the usefulness ofcertain characters into account, there is the strong possibility thatP. glans, P. maior, P. metarhiza, and P. tegere are actuallyconspecific. Lee (2001) also discussed this possibility and alsoincluded P. obovata to the list. We think that the apex morphologywith a papilla-like structure in combination with the truncated ant-apex characterizes P. obovata, and we therefore do not regard it assynonymous. Protaspis gemmifera and P. simplex are probablyalso synonymous. Perhaps, the conspecificity of these taxa shouldbe demonstrated with molecular methods before taxonomicchanges are proposed. Nonetheless, it is probable that the genuswill 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 havebeen observed but not formally described. For instance, Sournia(1986) reported a light micrograph of two cells that was referredto as Protaspis of unknown identity (p. 143, Fig. 51). Although theorganization of flagellar insertions and pseudopodia were notvisible, the overall morphology of this cell strongly suggests thatit is a Protaspis species with a thick cortex, anterior nucleus andlong ventral furrow.

Our phylogenetic analyses of P. grandis demonstrates a closerelationship between this species and C. longipes Schnepf et Kuhn2000 (Fig. 33). This result is consistent with ultrastructuralsimilarities between the two species. Like P. grandis, C. longipeshas a multilayered cell wall, a nucleus with a conspicuousnucleolus and condensed chromosomes, extrusomes of the samemorphology, pseudopodia that protrude through a preformed slitand flagella that sit within separated flagellar pits and emergethrough distinctive funnels (Schnepf and Kuhn 2000; Fig. 12–32).A multilayered cell wall, a nucleus with distinct areas ofcondensed chromatin and flagella emerging through funnels areultrastructural characters that have been used to diagnose thegenus Cryothecomonas (Thomsen et al. 1991; Table 1).

Nonetheless, there are clear ultrastructural differences betweenP. grandis and C. longipes, such as the organization of theflagellar insertion points, the abundance and distribution of con-densed chromosomes, the presence of a ventral furrow and therelative thicknesses of cell wall layers. The anterior flagellum isinserted apically and the posterior flagellum inserted subapicallyin C. longipes (Schnepf and Kuhn 2000), whereas both flagella areinserted 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 viewcontaining 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 4mm). 20. TEM showing an elongated Golgi body (dictyosome) containing seven stacked cisternae (Bar 5 0.25mm). 21. SEMshowing 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 1mm). 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 cellpreparing for division showing the anterior protrusion (double arrowhead) separating the replicated anterior flagella (arrows) and the replicated insertionfunnels (arrowheads) for the posterior flagella (Bar 5 1 mm).



reflect different swimming modes associated with the benthic andplanktonic lifestyles of most Protaspis and Cryothecomonasspecies, respectively. The nucleus of C. longipes is lobed, witha prominent nucleolus surrounded by heterochromatin aggregatednear the periphery of the nucleus (Schnepf and Kuhn 2000),whereas the heterochromatin in P. grandis is more abundant andevenly distributed in a non-lobed nucleus. Unlike P. grandis, theventral slit in C. longipes is not situated in a ventral furrow(Schnepf and Kuhn 2000). Key differences in the multilayeredcell wall of P. grandis, when compared with C. longipes are asfollows: (1) the coat is more stratified containing not only acolumnar mid-coat layer (like C. longipes) but also a deep coatlayer and a parallel superficial coat layer; (2) the intermediatelayer is significantly thicker and contains a vesicular layer em-bedded 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 thedescriptions presented for C. longipes (Schnepf and Kuhn2000), we were unable to rule out the possibility that the ‘‘outerlamina’’ in P. grandis actually represents the plasma membrane(Fig. 27–30).

These relatively modest differences in the basic charactersbetween these two species raise the question of whether P. grandisand C. longipes should be classified within two different genera.Moreover, is it sensible for us to generalize these observations toall members of both genera? If so, is it warranted to transfer P.grandis and related Protaspis species into the genus Cryotheco-monas or, conversely, to transfer C. longipes into the genusProtaspis (assuming Cryothecomonas is a junior synonym ofProtaspis)? Currently, we do not think that we can generalizeour ultrastructural results for P. grandis to be characteristic for allProtaspis species because there are no further TEM observationsfor the genus. It is highly likely that all species have a nucleuswith permanently condensed chromosomes, because the granularappearance of the nucleus is readily visible with light microscopy(which gave rise to previous classifications of Protaspis within thedinoflagellates and euglenids, see the Introduction). The posses-sion of a multilayered wall and flagellar insertion funnels shouldbe verified by ultrastructural investigations of the other Protaspisspecies. As circumscribed today, Protaspis seems to be a well-defined genus.

However, this is not the case for Cryothecomonas, and weargue that it is an artificial (polyphyletic) genus for the followingreasons. The type of flagellar insertion, the flagella motion and thesite of pseudopodia formation are different within the genus(Drebes et al. 1996; Schnepf and Kuhn 2000; Thomsen et al.1991). The type species Cryothecomonas armigera Thomsen et al.1991 has apically inserting isodynamic flagella separated by apapilla (Thomsen et al. 1991; Table 1 and Fig. 34). The pseudo-podia emerge from a slit (cytostome) located posterio-laterally(Thomsen et al. 1991; Table 1 and Fig. 34). All other species inthe genus that were described together with the type species,namely Cryothecomonas inermis, Cryothecomonas scybalophora,

and Cryothecomonas vesiculata, share these features (Thomsenet al. 1991; Table 1). Cryothecomonas aestivalis Drebes, Kuhn,and Schnepf, 1996 has apically inserting heterodynamic flagellaseparated by a papilla, and the pseudopodia emerge from theposterior cell pole (Drebes et al. 1996). Cryothecomonas longipeshas the anterior flagellum inserting apically, the posterior flagel-lum inserting subapically (heterodynamic), and a rarely noticeablepapilla (Schnepf and Kuhn 2000; Table 1 and Fig. 34). Thepseudopodia emerge from a ventral slit in the anterior cell half(Schnepf and Kuhn 2000; Table 1).

If we stress the importance of differences in the flagellarinsertion organization and patterns of motility and view them asdiagnostic characters at the generic level, then the genus Cryothe-comonas should be split into three genera, with C. aestivalis andC. longipes each representing a new genus. However, we hesitateto formally split the genus at this early stage, because there are nosequence data yet available for any Cryothecomonas species thatshares the same characters as the type. The SSU rDNA sequencefor the type species, C. armigera, should be added to the existingdataset before nomenclatural changes are recommended. None-theless, if the current members of Cryothecomonas were split intothree genera, then they would all be distinguishable from Protas-pis on the basis of flagellar insertion organization, with the new‘‘C. longipes-genus’’ being most closely related to it. Alterna-tively, it might be more appropriate to de-emphasize slightmodifications of the configuration of flagellar insertions (e.g.C. longipes versus P. grandis) and transfer all species of Cryothe-comonas into Protaspis, making the former a junior synonym ofthe latter. This would be the best approach to take if at some pointit is demonstrated that all of these species are intermingled withina robustly supported clade inferred from SSU rDNA sequences ora comparable molecular marker. Although there are no data forthe type species of Protaspis yet, it is likely that all of thesespecies whether placed in one or up to four different genera willshare the following features: (1) mitochondria with tubular cris-tae; (2) nucleus with permanently condensed chromosomes andconspicuous nucleolus; (3) multilayered cell wall outside of theplasma membrane; (4) feeding by means of pseudopodia; and(5) flagella emerging through funnels.

Molecular phylogeny and further expansion of the Cerco-zoa. Our phylogenetic analyses demonstrated that P. grandis isclosely related to C. longipes. The sequence from P. grandisrepresents only the third reference taxon (morphologicallydescribed species) within the Cryomonadida clade, which iscurrently comprised mainly of unidentified environmental se-quences (Fig. 33). A phylogenetic analysis excluding the shorterenvironmental sequences and including only morphologicallydescribed taxa within the Cercozoa (including P. grandis) ispresented elsewhere (Hoppenrath and Leander 2006). PreviousSSU rRNA phylogenies have shown that Cryothecomonas clus-ters robustly within the Cercozoa and have helped clarify therelative 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 throughwhich extrusomes are presumably discharged (arrows) and a mitochondrion with tubular cristae (m) (Bar 5 15mm). 28. High magnification transverseview of the cell wall showing seven distinct layers outside (above) the plasma membrane (pm): basal layer (bl), thick intermediate layer (il), vesicularlayer (vl) embedded within the intermediate layer, and outer lamina (ol) squeezed between the intermediate layer and the coat (co). The coat contain threesub-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 cellwall 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. Lowmagnification 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. Lowmagnification 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).


1996/97; Kuhn, Lange, and Medlin 2000). However, poor taxonsampling of heterotrophic amoeboflagellates that potentially be-long to the Cercozoa make it difficult to infer correct relationships

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

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), anteriorpapillae (arrowhead), and anterior protrusions (double arrowheads). Warts are shown on the surface of Protaspis tegere, Protaspis verrucosa, Protaspisgemmifera, and Protaspis simplex. Pseudopodia are shown emerging from the ventral furrow in Protaspis metarhiza and Protaspis obovata. Allillustrations are drawn approximately to scale. Redrawn after Skuja 1939 (Protaspis glans, Protaspis maior, Protaspis metarhiza), Skuja 1948 (Protaspisobovata), Lee 2001 (Protaspis tegere, Protaspis verrusosa, Protaspis gemmifera, Protaspis obliqua), Lee et al. 2005 (Protaspis simplex), Norris 1961(Protaspis tanyopsis), Schnepf and Kuhn 2000 (Cryothecomonas longipes), Drebes et al. 1996 (Cryothecomonas aestivalis).


Fig. 34. Gamma-corrected maximum likelihood tree (–ln L 5 8092.83, a5 0.28, number of rate categories 5 4) inferred using the GTR model ofsubstitution on an alignment of 54 small subunit (SSU) rDNA gene sequences and 1,006 sites. Numbers at the branches denote gamma-correctedbootstrap percentages using maximum likelihood—HKY (upper) and weighted neighbor-joining—GTR (middle). The lower number refers to Bayesianposterior probabilities—GTR. Black dots on branches denote bootstrap values and posterior probabilities of 100% in all analyses. The SSU rDNAsequence from Protaspis grandis n. sp. is highlighted in a black box and is closely related to Cryothecomonas longipes and other environmentalsequences within the larger Cryomonadida clade.


Tab

le1

.C

om

pari

son

of

morp

holo

gic

al

chara

cte

rst

ate

sin

desc

ribed

specie

sof

Pro

tasp

isan

dC

ryoth

ecom

onas.

Ch

ara

cte

rsP

.o

bli

qu

aP

.g

ran

dis

n.

sp.

P.

gla

ns

P.

gem

mif

era

P.

teg

ere

P.

verr

uco

sa

Fla

gell

ain

sert

ion

Subapic

al

Subapic

al,

funnel

Subap

ical

Subapic

al

Subapic

al

Subapic

al

Pro

tru

sio

nP

rotr

usi

on

No

No

No

No

Fla

gell

ar

len

gth

An

teri

ora

�0

.5–

0.7

5cl

�0

.5cl

�0

.5–

1�

cl

�cl

�1

–2�

cl

�cl

Po

steri

ora

�0

.5–

1.5

cl

�cl

1.2

–2�

cl

�1

.3–

3�

cl

�1

.5–

2.5�

cl

�1

.3–

2�

cl

Cell

shap

eR

ou

nd

-ov

al

Ov

al-

recta

ng

ula

rO

val

Ro

un

d-o

val

Ob

lon

g-o

vate

Ro

un

d-o

val

Cell

surf

ace

Sm

oo

thS

mo

oth

Sm

oo

thW

art

yS

mo

oth

or

wart

yF

ine

wart

sC

ell

flatt

enin

gD

ors

o-v

entr

al

Dors

o-v

entr

al

Dors

o-v

entr

al

Dors

o-v

entr

al

Dors

o-v

entr

al

Dors

o-v

entr

al

Cell

size

(mm

)L

en

gth

08

–3

23

2.5

–5

51

2–

30

08

–1

71

4–

25

09

–2

2W

idth

10

–2

72

0–

35

09

–15

08

–1

20

8–

14

8.5

–1

6A

nte

rio

rin

den

tati

on

Yes-

left

tom

ed

ian

No

No

No

No

No

Po

steri

or

ind

en

tati

on

Yes

No

No

No

No

No

Nu

cle

us

po

siti

on

An

teri

or

med

ian

Po

steri

or-

cen

tral

An

teri

or

or

cen

tral

late

ral

An

teri

or

med

ian

An

teri

or,

med

ian

or

rig

ht-

han

dsi

de

An

teri

or

med

ian

Nu

cle

us

shap

eS

ph

eri

cal

Sp

heri

cal

Sp

her

ical

Sp

heri

cal

Sp

heri

cal

Dis

co

idal

Nu

cle

ar

cap

No

No

No

No

/yesb

No

/yesb

No

/yesb

Condense

dchro

moso

mes

Unre

port

ed

Yes

Unre

port

ed

Unre

port

ed

Unre

port

ed

Unre

port

ed

Nucle

olu

sY

es

Yes

Unre

port

ed

Unre

port

ed

Unre

port

ed

Unre

port

ed

Ven

tral

furr

ow

/gro

ov

eP

ost

eri

or

half

,m

ed

ian

An

teri

or

half

,m

ed

ian

�F

ull

cell

len

gth

,m

ed

ian

Ind

isti

nct

�F

ull

cell

len

gth

,m

ed

ian�

Fu

llcell

len

gth

,m

ed

ian

Pse

ud

op

od

iaN

oY

es

Yes

Yes

Yes

Un

rep

ort

ed

Feed

ing

mo

de

Un

rep

ort

ed

Ph

ag

otr

oph

yP

hago

tro

ph

yP

hag

otr

oph

yP

hag

otr

op

hy

Un

rep

ort

ed

Body

scale

sU

nre

port

ed

No

Unre

port

ed

Unre

port

ed

Unre

port

ed

Unre

port

ed

Fla

gell

asc

ale

sU

nre

port

ed

No

Unre

port

ed

Unre

port

ed

Unre

port

ed

Unre

port

ed

Mo

vem

en

tG

lid

ing

Gli

din

gG

lid

ing

Gli

din

gG

lid

ing

Gli

din

gC

ell

wall

Yes

(lm

conly

)Y

es,

mult

ilayere

dU

nre

port

ed

Unre

port

ed

Unre

port

ed

Unre

port

ed

Extr

uso

mes

Unre

port

ed

Yes

Unre

port

ed

Unre

port

ed

Unre

port

ed

Unre

port

ed

Hab

itat

Ben

thic

Ben

thic

Ben

thic

,p

lan

kti

cB

en

thic

Ben

thic

,p

lan

kti

cB

en

thic

,p

lan

kti

c


Chara

cte

rsP

.m

aio

rP

.m

eta

rhiz

aP

.o

bo

va

taP

.si

mp

lex

P.

tan

yo

psi

s

Fla

gell

ain

sert

ion

Subapic

al

Subapic

al

Subapic

al

Subapic

al

Subapic

al

No

No

No

No

Pro

tru

sio

nF

lagell

ar

length

An

teri

ora

�cl

�0

.5–

0.3

cl

�cl

�0

.5–

1cl

�1

.2cl

Po

steri

ora

2�

cl

�cl

�cl

1–

3�

cl

�1

.3cl

Cell

shape

Oval

Oval,

post

eri

or

tip

Ovoid

(pear)

Round-o

val-

ovoid

Elo

ngate

Cell

surf

ace

Sm

ooth

Sm

ooth

Sm

ooth

Fin

ew

art

sin

som

eU

nre

port

ed

Cell

flatt

enin

gD

ors

o-v

entr

al

Dors

o-v

entr

al

Dors

o-v

entr

al

Dors

o-v

entr

al

Dors

o-v

entr

al

Cell

size

(mm

)L

en

gth

24

–4

02

8–

38

26

–4

04

.5–

25

28

–3

0W

idth

16

–3

01

6–

27

17

–2

50

2–

10

09

–1

1A

nte

rio

rin

den

tati

on

No

No

No

,p

ap

illa

No

No

Po

steri

or

ind

en

tati

on

No

No

No

,tr

un

cate

No

No

Nucle

us

posi

tion

Ante

rior

media

n,

Rig

ht

late

ral,

Ante

rior

Ante

rior,

Ante

rior

Ante

ro-l

ate

ral

Ante

rior–

centr

al

Ante

ro-l

ate

ral

Nucle

us

shape

Spheri

cal

Spheri

cal-

ovoid

Spheri

cal

Ovoid

Spheri

cal

Nucle

ar

cap

No

No

No

Yes,

inso

me

cell

sN

oC

on

dense

dch

rom

oso

mes

Un

rep

ort

ed

Un

rep

ort

ed

Yes

Un

rep

ort

ed

Un

rep

ort

ed

Nucle

olu

sY

es

Unre

port

ed

Unre

port

ed

Yes

Unre

port

ed

Ven

tral

furr

ow

/gro

ov

e�

cell

len

gth

,o

bli

qu

e�

cell

len

gth

,le

ftfr

om

med

ian

�cell

len

gth

,st

raig

ht

or

sig

mo

idS

hall

ow

,m

ed

ian

�2

/3C

ell

len

gth

Pse

ud

op

od

iaY

es

Yes

Yes

No

to

bse

rved

No

to

bse

rved

Fee

din

gm

od

eP

hag

otr

op

hy

Ph

ag

otr

op

hy

Ph

ag

otr

op

hy

Un

rep

ort

ed

Un

rep

ort

ed

Bo

dy

scale

sU

nre

po

rted

Un

rep

ort

ed

Un

rep

ort

ed

Un

rep

ort

ed

Un

rep

ort

ed

Fla

gell

asc

ale

sU

nre

port

ed

Unre

port

ed

Unre

port

ed

Unre

port

ed

Unre

port

ed

Mo

vem

en

tG

lid

ing

Gli

din

g,

swim

Sw

im,

rota

tio

nal

Gli

din

g,

wo

bb

lin

gC

ell

wall

Unre

port

ed

Unre

port

ed

Unre

port

ed

Unre

port

ed

Unre

port

ed

Ex

tru

som

es

Un

rep

ort

ed

Un

rep

ort

ed

Un

rep

ort

ed

Un

rep

ort

ed

Un

rep

ort

ed

Hab

itat

Ben

thic

,p

lan

kti

cP

lan

kti

cP

lan

kti

cB

en

thic

,p

lan

kti

cP

lan

kti

c

Tab

le1

.(C

on

tin

ued

).


Chara

cte

rsC

.lo

ng

ipes

C.

aest

iva

lis

C.

arm

igera

C.

inerm

isC

.sc

yb

alo

ph

ora

C.

vesi

cu

lata

Fla

gell

ar

inse

rtio

nA

pic

al

&su

bapic

al,

Apic

al,

funnels

,A

pic

al,

funnels

,A

pic

al,

funnels

,A

pic

al,

funnels

,A

pic

al,

funnels

,F

un

nels

,p

ap

illa

dP

ap

illa

Pap

illa

Pap

illa

Pap

illa

Pap

illa

Fla

gell

ar

length

An

teri

or

09

–1

5m

m1

5m

m1

–2�

cell

len

gth

1–2�

cell

len

gth

Un

rep

ort

ed

Un

rep

ort

ed

Po

steri

or

20

–2

4m

m,

fin

eh

air

s2

5m

m2

5m

m1

–2�

cell

length

Unre

port

ed

Unre

port

ed

Unre

port

ed

Hete

rodynam

icH

ete

rodynam

icIs

odynam

icIs

odynam

icU

nre

port

ed

Unre

port

ed

Cell

shape

Oval,

kid

ney

-shaped

Oblo

ng-o

val

Egg-s

haped

Egg-s

haped

Hig

hly

vari

able

Elo

ngate

dC

ell

surf

ace

Sm

ooth

Sm

ooth

Sm

ooth

Sm

ooth

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cercozoans (Ekelund, Daugbjerg, and Fredslund 2004; Kuhn,Medlin, and Eller 2004).

The Cercozoa was erected on the basis of molecular phyloge-netic data alone (Cavalier-Smith 1998a, b); no morphologicalfeature characterizes the whole group and the taxonomic diag-nosis is unusually broad (Cavalier-Smith 1998a, p. 232). Duringthe last 7 years, the identity and composition of the Cercozoa hasbeen 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 sisterclade to the Cryomonadida and was shown to comprise ebriids(Hoppenrath and Leander 2006). This resulted in the necessity toamend the diagnosis for the Cercozoa to include taxa with internalsiliceous skeletons. Our observations of P. grandis require addi-tional amendments to the diagnosis of the clade in order toaccommodate taxa with a rigid protein layer outside of the plasmamembrane (e.g. the cryomonads Protaspis and Cryothecomonas).These changes make the diagnosis for the clade even broader andmake the prospect of defining the clade on morphological groundsalone even more remote.

ACKNOWLEDGMENTS

We wish to thank W. J. Lee and D. J. Patterson for discussionson a part of the manuscript and help with the literature. We wouldlike to acknowledge discussions with Ø. Moestrup about thetaxonomic importance of flagellar insertions patterns and thankG. Eller who generously provided a provisional alignment ofsmall subunit rDNA sequences (‘‘Pseudopirsonia-alignment’’).This work was supported by a scholarship to M. Hoppenrath fromthe Deutsche Forschungsgemeinschaft (grant Ho3267/1-1) and bygrants to B. S. Leander from the National Science and Engineer-ing Research Council of Canada (NSERC 283091-04) and theCanadian Institute for Advanced Research. B. S. Leander is aScholar of the Canadian Institute for Advanced Research, Pro-gram 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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J. Eukaryot. Microbiol., 53(5), 2006 pp. 327–342 2006 … · Molecular Phylogenetic Position of Protaspis grandis n. sp. MONA HOPPENRATH and BRIAN S. LEANDER ... Patterson and Zo¨lffel