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Morphology and molecular phylogeny of a marine interstitial tetraflagellate with putative endosymbionts:… Chantangsi, Chitchai; Esson, Heather J; Leander, Brian S Jul 22, 2008

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ralssBioMed CentBMC MicrobiologyOpen AcceResearch articleMorphology and molecular phylogeny of a marine interstitial tetraflagellate with putative endosymbionts: Auranticordis quadriverberis n. gen. et sp. (Cercozoa)Chitchai Chantangsi*1, Heather J Esson2 and Brian S Leander1,2Address: 1Canadian Institute for Advanced Research, Program in Integrated Microbial Biodiversity, Department of Zoology, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada and 2Canadian Institute for Advanced Research, Program in Integrated Microbial Biodiversity, Department of Botany, University of British Columbia, Vancouver, BC, V6T 1Z4, CanadaEmail: Chitchai Chantangsi* - cchantan@interchange.ubc.ca; Heather J Esson - hjesson@interchange.ubc.ca; Brian S Leander - bleander@interchange.ubc.ca* Corresponding author    AbstractBackground: Comparative morphological studies and environmental sequencing surveys indicate that marinebenthic environments contain a diverse assortment of microorganisms that are just beginning to be explored andcharacterized. The most conspicuous predatory flagellates in these habitats range from about 20–150 μm in sizeand fall into three major groups of eukaryotes that are very distantly related to one another: dinoflagellates,euglenids and cercozoans. The Cercozoa is a diverse group of amoeboflagellates that cluster together inmolecular phylogenies inferred mainly from ribosomal gene sequences. These molecular phylogenetic studieshave demonstrated that several enigmatic taxa, previously treated as Eukaryota insertae sedis, fall within theCercozoa, and suggest that the actual diversity of this group is largely unknown. Improved knowledge ofcercozoan diversity is expected to help resolve major branches in the tree of eukaryotes and demonstrateimportant cellular innovations for understanding eukaryote evolution.Results: A rare tetraflagellate, Auranticordis quadriverberis n. gen. et sp., was isolated from marine sand samples.Uncultured cells were in low abundance and were individually prepared for electron microscopy and DNAsequencing. These flagellates possessed several novel features, such as (1) gliding motility associated with fourbundled recurrent flagella, (2) heart-shaped cells about 35–75 μm in diam., and (3) bright orange coloration causedby linear arrays of muciferous bodies. Each cell also possessed about 2–30 pale orange bodies (usually 4–5 μm indiam.) that were enveloped by two membranes and sac-like vesicles. The innermost membrane invaginated toform unstacked thylakoids that extended towards a central pyrenoid containing tailed viral particles. Although toour knowledge, these bodies have never been described in any other eukaryote, the ultrastructure was mostconsistent with photosynthetic endosymbionts of cyanobacterial origin. This combination of morphologicalfeatures did not allow us to assign A. quadriverberis to any known eukaryotic supergroup. Thus, we sequenced thesmall subunit rDNA sequence from two different isolates and demonstrated that this lineage evolved from withinthe Cercozoa.Conclusion: Our discovery and characterization of A. quadriverberis underscores how poorly we understand thediversity of cercozoans and, potentially, represents one of the few independent cases of primary endosymbiosiswithin the Cercozoa and beyond.Published: 22 July 2008BMC Microbiology 2008, 8:123 doi:10.1186/1471-2180-8-123Received: 20 March 2008Accepted: 22 July 2008This article is available from: http://www.biomedcentral.com/1471-2180/8/123© 2008 Chantangsi et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 16(page number not for citation purposes)BMC Microbiology 2008, 8:123 http://www.biomedcentral.com/1471-2180/8/123BackgroundMarine benthic environments contain a diverse assort-ment of microorganisms that are still just beginning to beexplored and characterized [1,2]. The challenges associ-ated with extracting and enumerating benthic microor-ganisms and the extreme variation of physical andchemical factors associated with the benthos have limitedour understanding of these ecosystems [2]. Nonetheless,both comparative morphological studies and environ-mental sequencing surveys have revealed a great deal ofmicroeukaryotic diversity within the interstitial spaces ofmarine sediments [3-16]. The most conspicuous preda-tory flagellates in these habitats range from about 20–150μm in size and fall into three major groups of eukaryotesthat are very distantly related to one another: dinoflagel-lates, euglenids and cercozoans.The Cercozoa is a large and diverse group of amoeboflag-ellates, with tubular mitochondrial cristae, that clustertogether in molecular phylogenies inferred mainly fromribosomal gene sequences (small and large subunitrDNA) [4,17-20]. Although a robust morphologicalsynapomorphy is currently lacking for the group, mem-bers of the Cercozoa do share novel molecular traits (i.e.molecular synapomorphies), such as the insertion of oneor two amino acid residues between the monomer tracksof highly conserved polyubiquitin genes [17]. Nonethe-less, molecular phylogenetic studies have demonstratedthat several enigmatic taxa, previously treated as Eukary-ota insertae sedis, fall within the Cercozoa, such as Allan-tion, Allas, Bodomorpha and Spongomonas [21];Cryothecomonas [22]; Ebria [23]; Gymnophrys and Lecythium[24]; Massisteria [25]; Metopion and Metromonas [4]; Prolep-tomonas [26]; and Protaspis [8]. Moreover, environmentalsequencing surveys have demonstrated several cercozoansubclades without clear cellular identities, suggesting thatthe actual diversity of this group is composed of thou-sands of uncharacterized lineages [4]. It must also beemphasized that morphological information from cerco-zoans, especially at the ultrastructural level, is largelyabsent from the literature. Accordingly, we characterizedthe ultrastructure and molecular phylogeny of a highlyunusual and rarely encountered tetraflagellate, Auranti-cordis quadriverberis n. gen. et sp. (Cercozoa), isolatedfrom sand samples collected in a marine tidal flat. Uncul-tured cells were individually isolated and prepared forDNA extraction (performed twice on different days, n = 5and n = 1), transmission electron microscopy (TEM, n = 2)and scanning electron microscopy (SEM, n = 25). Thisapproach enabled us to describe the ultrastructure ofintracellular pigmented bodies within A. quadriverberisthat are most likely photosynthetic endosymbiontsderived from cyanobacterial prey.ResultsGeneral morphology and behaviourAuranticordis quadriverberis was able to glide slowly usingfour tightly bundled flagella that were oriented posteri-orly. The cells of A. quadriverberis were also able to changeshape, albeit only slightly, and could be prominentlylobed, heart-shaped or ovoid (Figures 1A–F, H). In gen-eral, the cells had a narrower anterior apex and anexpanded posterior end and were composed of four majorlobes (L): L1, L2, L3, and L4 (Figure 1A). L1 was smallerthan other three lobes and was separated from L2, to theright, by a ventral depression (vd) and separated from L4,to the left, by a ventral groove (gr) that contained the fourrecurrent flagella (Figure 1A). Apart from differences incell shape and the effects of cell plasticity, there was alsovariation in the size of different individuals, ranging from35–75 μm in diam. (n = 65). The cells were conspicuouslyorange in color, caused mostly by the presence of lineararrays of tiny orange muciferous bodies that were distrib-uted over the entire surface of the cell (Figures 1A–B).Microscopical observations indicated that these bodiessecrete sticky mucilage when the cells are disturbed, sug-gesting that the bodies function for adhesion to the sub-stratum. TEM micrographs showed that the muciferousbodies were small compartments (780 nm in diam.) posi-tioned underneath the cell membrane and filled withamorphous material that was secreted as mucilaginousstrands (Figures 2D, 3B–C). The surface of A. quadriver-beris was also corrugated and consisted of over 80 longitu-dinal ridges that spanned from the anterior apex to theposterior end (Figures 2A–C). The grooves between theridges contained numerous tiny pores through which themucilage from the muciferous bodies was secreted (Figure2C). TEM sections through the cell surface also demon-strated a single row of microtubules positioned beneatheach ridge (Figure 3E). No test or cell wall was present.The four flagella of A. quadriverberis originated from ananterior flagellar pocket and nestled tightly within theventral groove, making them nearly invisible under thelight microscope (Figures 1B, 1D–E, 1G, 2A–B, 2E, 4F).Electron microscopy demonstrated that the flagella werearranged in two pairs and covered with flagellar hairs ormastigomenes (Figure 2E). Except for very slight differ-ences in length, all four flagella were morphologicallyidentical and slightly longer than the cell (Figures 1B, D–E). The flagella were also homodynamic and associatedwith gliding motility along the substratum. Pseudopodiawere not observed.Main cytoplasmic componentsAuranticordis quadriverberis contained a large nucleus (15–20 μm in diam.) situated in the anterior region of the cellPage 2 of 16(page number not for citation purposes)(Figures 3A, 3D). Although the position of the nucleus inliving specimens cannot be readily seen under the lightBMC Microbiology 2008, 8:123 http://www.biomedcentral.com/1471-2180/8/123Page 3 of 16(page number not for citation purposes)Light micrographs (LM) of Auranticordisquadriverberis n. gen. et sp. showing cell color, main cytoplasmic components, and varia-tion in cell sh eFi ure 1Light micrographs (LM) of Auranticordisquadriverberis n. gen. et sp. showing cell color, main cytoplasmic components, and varia-tion in cell shape. A. Differential interference contrast (DIC) image focused on rows of longitudinally arranged orange mucifer-ous bodies (arrowhead), the ventral groove (double arrowhead), lobe 1 (L1), a ventral depression (vd), L2, L3, and L4. B. An inverted heart-shaped cell with visible flagella (arrow) emerging from the posterior region of the ventral groove. C. A flattened cell showing larger pale orange bodies (putative primary endosymbionts, arrowheads) distributed in the anterior end of the cell. D. DIC image showing the position of the ventral groove (double arrowhead) with flagella (arrow) relative to a prominent L1 and L4. E. Phase contrast micrograph demonstrating the distal end of the flagella emerging from the ventral groove. F. DIC micrograph showing black bodies (asterisk) accumulated at the anterior end of the cell and two pale orange bodies (putative primary endosymbionts, arrowheads). G. A squashed cell showing the anterior nucleus (N) and flagella (arrow). H. DIC micro-graph showing a cell with prominent lobes. I. A squashed cell showing variation in the shape and size of the pale orange bodies (putative primary endosymbionts, arrowheads). (A-I, Bar = 10 μm).BMC Microbiology 2008, 8:123 http://www.biomedcentral.com/1471-2180/8/123microscope, the nucleus is visible in compressed cells as a nucleoli (Figures 3A, 3D, 4D–E). The nucleus was pointedScanning electron micrographs (SEM) of Auranticordis quadriverberis n. gen. et spFigure 2Scanning electron micrographs (SEM) of Auranticordis quadriverberis n. gen. et sp. A. An anterior view of the cell showing the anterior apex (arrowhead), ventral groove (double arrowhead) and flagella (arrow) (Bar = 10 μm). B. A higher magnification view of the anterior end of the cell (arrowhead) showing the flagella (arrow) within the ventral groove (double arrowhead) (Bar = 2 μm). C. High magnification view of the ridges showing several tiny pores (arrowheads) in the grooves (Bar = 1 μm). D. High magnification view of secreted mucus (arrowheads) (Bar = 0.5 μm). E. High magnification view of the ventral groove showing all four flagella (arrows) bundled together and covered in hairs (Bar = 0.5 μm).Page 4 of 16(page number not for citation purposes)comparatively clear area (Figures 1G, 1I). TEM sectionsdemonstrated the nuclear envelope and a few prominentat the anterior end and was connected to a striated bandnear the basal bodies and microtubular roots (Figures 3D,BMC Microbiology 2008, 8:123 http://www.biomedcentral.com/1471-2180/8/123Page 5 of 16(page number not for citation purposes)Transmission electron micrographs (TEM) of Auranticordis quadriverberis n. gen. et spFigure 3Transmission electron micrographs (TEM) of Auranticordis quadriverberis n. gen. et sp. A. Low magnification view showing the main cellular components: black bodies (b), nucleus (N), pale orange bodies (putative primary endosymbionts, PE), a degraded PE (double arrowhead) surrounded by sac-like vesicles (asterisk), surface ridges (arrows), and the ventral depression (vd) (Bar = 10 μm). B. Section through the surface showing a row of muciferous bodies (arrowheads) containing (orange) amorphous material. Each muciferous body is about 500–900 nm in diameter (Bar = 0.5 μm). C. High magnification view of muciferous bodies (arrowheads) and secreted mucus (arrow) (Bar = 0.5 μm). D. Section through the anterior region of the cell showing black bodies (b), the flagellar pocket (double arrowhead), four flagella (arrows), a nucleolus (n), a pointed nucleus (N), and the ventral groove (gr) (Bar = 5 μm). E. High magnification section through the surface ridges (arrow) showing underlying micro-tubules (arrowhead) and muciferous bodies (double arrowheads) (Bar = 0.5 μm). An inset showing a magnified view of a sur-face ridge (arrow) with a row of microtubules underneath (arrowhead) (Bar = 0.5 μm). F. Transverse section showing all four flagella within a flagellar pocket (arrowhead) near the nuclear anterior projection (N) (Bar = 1 μm).BMC Microbiology 2008, 8:123 http://www.biomedcentral.com/1471-2180/8/123Page 6 of 16(page number not for citation purposes)Transmission electron micrographs (TEM) of Auranticordis quadriverberis n. gen. et sp., showing different cytoplasmic compo-nentFigure 4Transmission electron micrographs (TEM) of Auranticordis quadriverberis n. gen. et sp., showing different cytoplasmic compo-nents. A. High magnification TEM showing a vacuolated cytoplasm (arrowheads) and fibrous material (fs) distributed beneath the cell periphery (Bar = 0.5 μm). B. High magnification view of the black inclusions (arrowheads) (Bar = 2 μm). C. An ingested bacterium found within cytoplasm of A. quadriverberis (Bar = 0.25 μm). D. A section through the nucleus (N) showing nucleoli (arrowheads) and an invaginated area (double arrowhead) (Bar = 2 μm). E. High magnification TEM showing the nuclear enve-lope (arrow), the nucleus (N), and a striated band (double arrowhead) positioned between the nuclear tip and a microtubular root (arrowhead) (Bar = 0.5 μm). F. Tangential section through the flagella (arrowheads) lying within the ventral groove (gr) (Bar = 1 μm). G. A putative mitochondrion positioned near the cell periphery (Bar = 0.2 μm). An inset showing two putative mitochondria (Bar = 0.5 μm). H. TEM showing lipid globules (lg) near the posterior part of the cell (Bar = 1 μm). I. High mag-nification view of a Golgi apparatus (Bar = 0.5 μm).BMC Microbiology 2008, 8:123 http://www.biomedcentral.com/1471-2180/8/1234D–E). Moreover, bundles of (non-microtubular) fibrousmaterial were also observed within the cytoplasm near thecell periphery (Figure 4A).The cells of A. quadriverberis also contained an accumula-tion of black material near the anterior part of the cell,lipid globules and Golgi bodies (Figures 1C, 1F, 1I, 4H–I). Although mitochondria with tubular cristae were notdefinitively observed, several elongated bodies that werehighly reminiscent of acristate mitochondria were foundnear the periphery of the cell (Figures 3A, 4G). The cellsalso contained 2–30 pale orange bodies that were variablein shape and usually about 4–5 μm in diam.; however,some of these bodies were 14 μm long (Figures 1C, 1F, 1I,3A, 5A–G, 6). The pale orange bodies were distributedthroughout the cell, but were most abundant in the ante-rior region of the cell. Each pale orange body was envel-oped by two tightly pressed inner membranes andsurrounded by sac-like vesicles (Figures 5A, 5C, 5F). Theinnermost membrane invaginated into the lumen of thebody and formed several unstacked thylakoids around theperiphery (Figures 5A–C, 5E). The sac-like vesicles occa-sionally butted together to form perpendicular partitionsoutside of the two inner membranes (Figure 5F). The cen-tral core of the pale orange bodies was devoid of mem-branes and contained a central electron dense regioncontaining tailed viral particles (Figures 5D, 5G).Molecular phylogenetic position of auranticordisPhylogenetic analyses of a 69-taxon dataset representingall major groups of eukaryotes showed A. quadriverberisbranching within the Cercozoa with very strong statisticalsupport (data not shown). This cercozoan clade, com-prised of Chlorarachnion reptans, Cryothecomonas aestivalis,C. longipes, Ebria tripartita, Euglypha rotunda, Heteromitaglobosa and A. quadriverberis, was strongly supported inboth maximum likelihood (ML) and Bayesian analyses(ML boostrap = 100 and Bayesian posterior probabilities= 1.00; data not shown). A more comprehensive analysisof 981 homologous positions in 126 cercozoan SSUrDNA sequences, including several shorter environmentalsequences, placed A. quadriverberis near Pseudopirsoniamucosa (a parasitic nanoflagellate of diatoms) and twounidentified cercozoans with 1.00 Bayesian posteriorprobabilities (data not shown). Accordingly, we per-formed phylogenetic analyses of 1,571 positions in 32cercozoan taxa that excluded the shortest environmentalsequences and included the closest relatives of A. quadriv-erberis in the 126-taxon alignment.Figure 7 illustrates the phylogenetic analyses of the 32-taxon dataset. Like in the analyses of 126 taxa, the two dif-ferent isolates of A. quadriverberis clustered with twomental sequence AB252755 was recovered with aposterior probability of 1.00 and 73% PhyML bootstrapvalue. A more inclusive clade consisting of A. quadriver-beris, P. mucosa and environmental sequences AB252755and AB275058 received high statistical support (posteriorprobability of 1.00 and PhyML bootstrap value of 97%)(Figure 7). Members of this clade also shared a derivedmolecular character within the context of 160 cercozoansequences covering representatives from all known cerco-zoan subclades: namely, the substitution of cytosine (C)for thymine (T) at position 324 (with reference to thecomplete SSU rDNA sequence of Cercomonas sp.; Gen-Bank accession no. AF411266, culture ATCC PRA-21) inHelix 12, based on the predicted secondary structure ofthe SSU rRNA gene in Palmaria palmata [27].DiscussionComparative morphologyThe distinctly orange color of A. quadriverberis sets theseflagellates apart from other organisms living in the samebenthic environment. To our knowledge, similar organ-isms have not been recorded previously [3,9-12,28]; how-ever, the orange color of A. quadriverberis is mostreminiscent of the anoxic euglenozoan Calkinsia aureus[29].The presence of four recurrent flagella in A. quadriverberisis another distinctive feature. Most cercozoans possesstwo flagella, although Cholamonas cyrtodiopsidis also hasfour flagella that are inserted subapically [30,31]. The flag-ella of C. cyrtodiopsidis form two symmetrical pairs com-prising one long and one stubby flagellum [30,31]. Thisflagellar organization differs from A. quadriverberis, whichhas two pairs of tightly bundled flagella originating fromthe same flagellar reservoir. Cholamonas cyrtodiopsidis wasassigned to the Cercomonadida due to possession of amicrobody and kinetid architecture that is similar to somespecies of Cercomonas [30,31]. Although both A. quadriv-erberis and C. cyrtodiopsidis possess four flagella, this char-acter state is unlikely to be synapomorphic for thesespecies: A. quadriverberis inhabits marine sand, whereas C.cyrtodiopsidis inhabits the intestines of diopsid flies [30].Moreover, the distinctive features present in one speciestend not to be shared by the other (e.g. the paranuclearbodies found in C. cyrtodiopsidis are not present in A. quad-riverberis). Because the phylogenetic position of C. cyrtodi-opsidis has not yet been evaluated with molecularphylogenetic data, our ability to infer the evolution of thetetraflagellated state within the Cercozoa is limited.The flagella of A. quadriverberis are covered by hairs, andalthough this stands in contrast to the smooth flagelladescribed in most other cercozoans, such as CercomonasPage 7 of 16(page number not for citation purposes)uncultured eukaryotes and P. mucosa (Figure 7). A subc-lade consisting of A. quadriverberis, P. mucosa and environ-and Proleptomonas [31], the hairs could be homologous tothose described in the predatory soil-dwelling flagellateBMC Microbiology 2008, 8:123 http://www.biomedcentral.com/1471-2180/8/123Page 8 of 16(page number not for citation purposes)Transmission electron micrographs (TEM) showing the ultrastructure of putative primary endosymbionts in Auranticordis quad-riverberis n. gen. et spFigure 5Transmission electron micrographs (TEM) showing the ultrastructure of putative primary endosymbionts in Auranticordis quad-riverberis n. gen. et sp. A. Low magnification TEM showing four putative endosymbionts, each surrounded by sac-like vesicles (sc) defined by an outer membrane (Bar = 2 μm). B. High magnification TEM showing two enveloping inner membranes (arrowheads) and thylakoids (arrows) that are continuous with the innermost enveloping membrane (Bar = 0.2 μm). C. TEM showing the thylakoids, the sac-like vesicle (sc), and a cleavage furrow indicative of division (arrowheads) (Bar = 0.5 μm). D. High magnification TEM showing the central core of an endosymbiont containing viral particles (arrowheads) (Bar = 0.5 μm). E. High magnification TEM showing a pronounced invagination of the innermost enveloping membrane (arrowhead) (Bar = 0.5 μm). F. High magnification TEM showing the membrane (arrowheads) that defines the sac-like vesicle (sc) and the two inner-most enveloping membranes (double arrowheads) (Bar = 0.2 μm). G. TEM showing viral particles (arrowhead) consisting of a polygonal head and tail, and positioned within the core of an endosymbiont (Bar = 0.5 μm). An inset showing a complete tailed viral particle (Bar = 0.2 μm).BMC Microbiology 2008, 8:123 http://www.biomedcentral.com/1471-2180/8/123Aurigamonas solis [16,32]. The four flagella of A. quadriver-beris were also recurrent and homodynamic during glid-ing motility, which is unlike the heterodynamic flagella ofmost other interstitial cercozoans (e.g. Cercomonas, Heter-omita, Katabia, Proleptomonas, and Protaspis) [8,31]. Thegliding cells of A. quadriverberis were plastic and capable ofslow changes in shape that was somewhat similar to thatfound in euglenids [33]. This plasticity is probably gener-ated by the row of microtubules locating underneath thecell membrane (Figure 3E).The nucleus of A. quadriverberis is difficult to see in livingcells, which is also unlike most other cercozoans (e.g.Aurigamonas, Cercomonas, Ebria, Euglypha, Heteromita,Protaspis, Thaumatomastix, and Thaumatomonas)[8,10,16,23,34]. The bloated shape of the cell and thenucleus. The ultrastructure of the nucleus is similar to thatof other cercozoans (e.g. contained several nucleoli)[8,16,35-37]; however, A. quadriverberis lacked perma-nently condensed chromosomes like those found in Cryo-thecomonas, Ebria, and Protaspis [8,16,23,35,37,38]. Theshape of the nucleus in A. quadriverberis was indented atone side, a feature also noticed in the nucleus of Protaspisgrandis [8], and had a prominent anterior projection ori-ented towards the flagellar pocket. An anterior projectionwas also observed in the nucleus of Cercomonas; in bothgenera, the anterior projection was associated with abroad striated band and the ventral (posterior) roots ofthe anterior and posterior flagella (VP) [31,36]. However,the characteristic microtubular cone present in Cer-comonas [31,36] was not observed in A. quadriverberis.The cytoplasm of A. quadriverberis contained lipid glob-ules, Golgi bodies and muciferous bodies. The muciferousbodies were compartments organized in linear arrays andfilled with an amorphous matrix that appeared brightorange under the light microscope. Extrusomes like thesehave also been reported in C. armigera as a minute periph-eral concavities filled with a homogeneous matrix [37].Other types of extrusomes that have been found in differ-ent cercozoan species, such as trichocysts, microtoxicysts,kinetocysts and osmiophilic bodies, [8,31,36], wereabsent in A. quadriverberis. The lipid globules varied con-siderably in size and were most abundant in the posteriorregion of A. quadriverberis. These globules were reminis-cent of those described in Protaspis [8]. Although themode of feeding in A. quadriverberis was not clearlyobserved, evidence of ingested bacteria was observedwithin its cytoplasm (Figure 4C).The cytoplasm of A. quadriverberis was highly vacuolatedand looked similar to the cytoplasm described in Cryothe-comonas armigera and Protaspis grandis [8,37]. The anteriorpart of the cell, however, contained black bodies similarto those that have been observed in other distantly relatedeukaryotes, such as some semi-anoxic euglenids and cili-ates. Moreover, distinct mitochondria with tubular cristae,which are characteristic of other cercozoans, were notfound in A. quadriverberis. Putative mitochondria were,however, observed around the cell periphery (Figure 4G),and the lack of cristae in these organelles reflects eitherdegenerate mitochondria associated with a low-oxygenenvironment or fixation artifact [39]. The size of the puta-tive mitochondria ranged between 135–185 nm long,which is smaller than the mitochondria described in mostcercozoans. For example, the mitochondria of Auriga-monas solis are about 630 nm [16], the mitochondria ofCercomonas are about 485 nm [36], the mitochondria ofCryothecomonas longipes are about 280 nm [40], and theA schematic line drawing of Auranticordis quadriverberis n. gen. et spFigure 6A schematic line drawing of Auranticordis quadriverberis n. gen. et sp. The line drawing was constructed from light micro-graphs and showing a lobed cell, rows of tiny orange mucifer-ous bodies (small circles), four flagella within ventral groove, a ventral depression (lightly stippled area to the left of the flagella), and four putative primary endosymbionts (large shaded circles).Page 9 of 16(page number not for citation purposes)dense distribution of minute orange muciferous bodiesthat subtend the entire surface of the cell obscured themitochondria of P. grandis are about 500 nm [8].Although the implementation of fluorescent stains, likeBMC Microbiology 2008, 8:123 http://www.biomedcentral.com/1471-2180/8/123Page 10 of 16(page number not for citation purposes)Maximum likelihood (ML) tree (-ln L = 10139.70214) inferred from 32 SSU rDNA sequences, 1,571 unambiguously aligned sites and a GTR+I+G+8 model of nucleotide substitutionsFigure 7Maximum likelihood (ML) tree (-ln L = 10139.70214) inferred from 32 SSU rDNA sequences, 1,571 unambiguously aligned sites and a GTR+I+G+8 model of nucleotide substitutions. Numbers above the branches denote PhyML bootstrap percentages, and numbers below the branches denote Bayesian posterior probabilities. Black circles denote PhyML bootstrap percentages and posterior probabilities of 100% and 1.00, respectively. Line drawings were modified from the following sources: Massisteria marina [10], Pseudodifflugia gracilis [70], Cryothecomonas sp. [71], Ebria tripartita [72], Cercomonas sp. [73], Euglypha alveolata [74], Heteromita globosa [34], Thaumatomonas lauterborni [75], and Pseudopirsonia sp. [76]. The asterisk next to sequence [Gen-Bank:DQ388459] was derived from an environmental sequencing survey and was listed in GenBank as the dinoflagellate Exuvi-aella pusilla by Lin et al. [77].BMC Microbiology 2008, 8:123 http://www.biomedcentral.com/1471-2180/8/123Mitotracker, could help establish the identity of thesestructures [41], this approach is limited by the scarcity ofthese organisms in natural environments and the unpre-dictability of finding them in our samples.Putative primary endosymbiontsSeveral light orange bodies about 4–14 μm in diam. weredistributed within the cell and were especially abundanttowards the anterior end of the cell. Although theultrastructure of these pigmented bodies is novel, thepresence of thylakoid-like membranes and a central spacecontaining a densely stained inclusion is consistent withthree possible identities that differ by the degree of inte-gration with the host cell: (1) the bodies are ingested(photosynthetic) prey cells that are in the earliest stages ofbeing degraded, (2) the bodies are transient photosyn-thetic endosymbionts that are continuously replenishedby kleptoplasty, or (3) the bodies are permanently inte-grated photosynthetic endosymbionts (i.e. plastids). Theplausibility of each of these hypotheses is addressedbelow.The orange color of these bodies is reminiscent of theplastids in some microalgae, such as dinoflagellates anddiatoms that occupy the same habitats as A. quadriverberis.However, neither dinoflagellate theca nor diatom frus-tules were found associated with these bodies in any TEMsections, and the ultrastructure of the bodies was very dif-ferent from the known ultrastructural diversity in the plas-tids of diatoms and dinoflagellates. Some cyanobacteriaare known to have pale orange coloration that is similarto the orange bodies within A. quadriverberis [42]. Theseorange bodies were surrounded by two tightly com-pressed inner membranes and sac-like vesicles. Whereastypical food bodies show degrees of being digested by cel-lular enzymes, nearly all of the pigmented bodiesobserved were completely intact in all of the cells weobserved (n = 70), suggesting that they are constant fix-tures of the host cell cytoplasm.Primary endosymbiosis, involving a photosyntheticprokaryote within a eukaryotic cell, results in three sur-rounding membranes: two cyanobacterial inner mem-branes and a third, outer phagosomal membrane. Greenalgae/land plants, red algae, and glaucophytes possess pri-mary plastids [43-45]. Two membranes surround the plas-tids of green algae and red algae, and the third outerphagosomal membrane is inferred to have been lost [43-46]. Secondary endosymbiosis occurs through the engulf-ment, integration and maintenance of either a green orred alga by a predatory eukaryote. This process producedthe plastids of cryptomonads, haptophytes, strameno-piles, dinoflagellates, apicomplexans, and euglenids [43-rarachniophytes have secondary plastids derived fromgreen algae [45,47] and (2) Paulinella chromatophora hasprimary plastids derived from cyanobacterial prey [48-50].Like in Paulinella and the cyanelles of glaucophytes, theultrastructure of the pigmented bodies within A. quadriv-erberis is most consistent with the ultrastructure of free-liv-ing cyanobacteria, suggesting an independent primaryendosymbiotic origin [44,48-52]. For instance, TEM sec-tions through the pigmented bodies demonstrated amode of division that is similar to division described inthe cyanelles of Cyanophora paradoxa [53] (Figure 5C).Moreover, the thylakoids in the endosymbionts of P. chro-matophora, the cyanelles of glaucophytes, and coccoidphotosynthetic cyanobacteria are unstacked and arrangedconcentrically around the periphery of the cell [48,54,55].A similar arrangement was observed in the pigmentedbodies of A. quadriverberis (Figure 5A–C), although themajority of the thylakoids projected inward towards thecore of the body. The central area within the pigmentedbodies of A. quadriverberis resembled the pyrenoids in thecyanelles of Glaucocystis nostochinearum [55].The thylakoid-free core of the pigmented bodies also con-tained polygonal viral particles. TEM sections throughthese particles demonstrated complete tailed phages sim-ilar to those known to infect cyanobacteria [56-58] (Fig-ure 5G). Viral particles similar to those described in thepigmented bodies of A. quadriverberis have also beendescribed in the same region in the plastids of othereukaryotes, such as the "polyhedral bodies" in the pri-mary endosymbionts of P. chromatophora [48], thecyanelles of the glaucophyte Gloeochaete wittrockiana [55],and the free-living photosynthetic cyanobacterium Nostocpunctiforme [54]. Two other important characters that havebeen used to infer a cyanobacterial origin for primaryplastids are: (1) the presence of phycobilisomes and (2)the presence of a peptidoglycan wall [48,49,51]. However,as previously mentioned, neither phycobilisomes nor apeptidoglycan layer was present in the orange bodies in A.quadriverberis.ConclusionOur characterization of A. quadriverberis n. gen. et sp. dem-onstrates several novel features within the Cercozoa, suchas four homodynamic flagella, densely distributed linearrows of orange muciferous bodies, and putative endosym-bionts with an enigmatic overall structure. The discoveryof this highly distinctive lineage underscores how poorlywe understand the actual cellular diversity of cercozoansand, potentially, represents one of the few independentcases of primary endosymbiosis within the Cercozoa andPage 11 of 16(page number not for citation purposes)45]. Two different lineages of cercozoans have independ-ently acquired plastids through endosymbiosis: (1) chlo-beyond. Although endosymbioses are known to haveoccurred many different times independently, the trans-BMC Microbiology 2008, 8:123 http://www.biomedcentral.com/1471-2180/8/123formation of endosymbionts into organelles is consideredto be much less common [59]. In order to more confi-dently infer the origin of the pigmented bodies in A. quad-riverberis, experiments involving autofluorescence and theamplification of plastid molecular markers (e.g. 16SrDNA and psb genes) could be performed [50]. These stud-ies will be hampered mainly by the scarcity and unpredict-ability of finding these cells in natural samples.Nonetheless, additional studies on A. quadriverberis andits putative endosymbionts will enable us to better under-stand the extent of endosymbiosis across the tree ofeukaryotes and the convergent processes associated withthe establishment and integration of endosymbiontswithin eukaryotic cells.Taxonomic descriptionsTaxonomic treatment for Auranticordis quadriverberisPhylum Cercozoa [60]Genus Auranticordis gen. nov. Chantangsi, Esson and Leander 2008DiagnosisUninucleate tetraflagellates; four recurrent flagellainserted subapically and bundled together within a ven-tral longitudinal groove; all flagella about one cell length;cell shapes are prominently lobed, ovoid or heart-shaped;nucleus at anterior end of cell, with nucleoli; no cell wallor test; minute orange muciferous bodies distributed inlinear arrays over the entire cell; cytoplasm with paleorange pigmented bodies, usually concentrated at theanterior end; corrugated cell surface; black inclusions usu-ally present at anterior part of the cell; locomotion byslow gliding; cell deformations possible; marine habitat.Type speciesAuranticordis quadriverberis.EtymologyLatin aurantium, n. orange; L. cordis, n. heart. The genericname reflects two characteristic features of this taxon:orange cell coloration and inverted heart-shaped cells.Species Auranticordis quadriverberis spec. nov. Chantangsi, Esson and Leander 2008DescriptionCell shape ovoid, prominently lobed or inverted heart-shaped; cell size 35–75 μm long, 25–70 μm wide; fourhomodynamic flagella, inserted subapically and bundledwithin a ventral longitudinal groove; anterior nucleuswith nucleoli; bright orange coloration caused by linearrows of minute orange muciferous bodies; corrugated cellsurface with about 80 longitudinal ridges; no cell wall ortest; cytoplasm with 2–30 pale orange pigmented bodies;sequences [GenBank:EU484393 and Gen-Bank:EU484394].Type localityTidal sand-flat at Spanish Banks, Vancouver, BritishColumbia, Canada. The specimen was found duringMarch and May, 2007.HapantotypeBoth resin-embedded cells used for TEM and cells on goldsputter-coated SEM stubs have been deposited in theBeaty Biodiversity Research Centre (Marine InvertebrateCollection) at the University of British Columbia, Van-couver, Canada.IconotypeFigures 1B, 1F, 1H and 6.Type localitySpanish Banks, Vancouver, BC, Canada (39°28' N, 74°15'W).HabitatMarine sand.EtymologyThe etymology for the specific epithet, Latin quattuor, four;L. verberis, n. whip. The specific epithet reflects the pres-ence of four flagella.MethodsSampling and light microscopy (LM)Sand samples were collected from Spanish Banks, Van-couver, BC, Canada in March 2007. Organisms wereextracted from the sand samples through a 48 μm meshusing a melted seawater-ice method described by Uhlig[61]. Briefly, 2–3 spoons of sand samples were placed intoan extraction column wrapped with a 48 μm mesh. Twoto three seawater ice cubes were then put on top of thesand samples and left to melt over several hours. Theorganisms of interest were separated through the meshand concentrated in a Petri dish that was filled with sea-water and placed underneath the extraction column. ThePetri dish containing the organisms was then screenedusing a Leica DMIL inverted microscope. Cells were indi-vidually isolated and placed on a slide for light micros-copy using phase contrast and differential interferencecontrast (DIC) microscopy with a Zeiss Axioplan 2 imag-ing microscope connected to a Leica DC500 color digitalcamera.Scanning electron microscopy (SEM)Twenty-five cells of Auranticordis quadriverberis were indi-Page 12 of 16(page number not for citation purposes)black inclusions usually present at anterior part of the cell;locomotion by slow gliding. Small subunit rRNA genevidually isolated and placed into a small container cov-ered on one side with a 10-μm polycarbonate membraneBMC Microbiology 2008, 8:123 http://www.biomedcentral.com/1471-2180/8/123filter (Corning Separations Div., Acton, MA, USA). Thesamples were pre-fixed in the container with OsO4 vaporfor 30 min at room temperature and subsequently post-fixed for 30 min with a mixture of 8% glutaraldehyde and4% OsO4, giving a final concentration of 2.5% glutaralde-hyde and 1% OsO4. The organisms were then washedthree times in filtered seawater to remove the fixative anddehydrated through a graded series of ethanol. Dehy-drated samples were critical point dried with CO2 using aTousimis Samdri 795 CPD (Rockville, MD, USA). Driedfilters containing the cells were mounted on aluminumstubs and then sputter coated with gold (5 nm thickness)using a Cressington high resolution sputter coater (Cress-ington Scientific Instruments Ltd, Watford, UK). Thecoated cells were viewed under a Hitachi S4700 scanningelectron microscope.Transmission electron microscopy (TEM)Two individual cells of Auranticordis quadriverberis wereprepared separately. Each cell was pre-fixed with 2% (v/v)glutaraldehyde (in unbuffered seawater) at room temper-ature for 1 h. Cells were then washed three times in fil-tered seawater and post-fixed with 1% (v/v) OsO4 (inunbuffered seawater) for another 1 h at room tempera-ture. Fixed cells were then washed three times in filteredseawater and were dehydrated through a graded series ofethanol. Infiltration was performed with acetone-resinmixtures (acetone, 2:1, 1:1, 1:2, Epon 812 resin) and indi-vidually flat embedded in Epon 812 resin. The resin con-taining the cell(s) was polymerized at 65°C for one dayand sectioned with a diamond knife on a Leica EM-UC6ultramicrotome. The sections were collected on copper,formvar-coated slot grids and stained with uranyl acid andlead citrate (Sato's lead method) [62,63]. TEM micro-graphs were taken with a Hitachi H7600 transmissionelectron microscope.DNA extraction and PCR amplificationFive cells were individually isolated and washed threetimes in autoclaved seawater. DNA was extracted usingthe protocol provided in the Total Nucleic Acid Purifica-tion kit by EPICENTRE (Madison, WI, USA). Polymerasechain reaction (PCR) was performed in a thermal cyclerusing puReTaq Ready-To-Go PCR beads (GE HealthcareBio-Sciences, Inc., Québec, Canada). The forward (PF1: 5'-GCGCTACCTGGTTGATCCTGCC-3') and reverse (R4: 5'-GATCCTTCTGCAGGTTCACCTAC-3') primers for ampli-fying SSU rDNA were added into the tube with the finalreaction volume of 25 μl. The thermal cycler was pro-grammed as follows: hold at 94°C for 4 min; 5 cycles ofdenaturation at 94°C for 30 sec, annealing at 45°C for 1min, and extension at 72°C for 105 sec; 35 cycles of dena-turation at 94°C for 30 sec, annealing at 55 °C for 1 min,were separated by agarose gel electrophoresis, cleanedusing the UltraClean™ 15 DNA Purification Kit (MO BIOLaboratories, Inc., CA, USA). The cleaned DNA wascloned into pCR2.1 vector using the TOPO TA Cloning®kits (Invitrogen Corporation, CA, USA). Plasmids with thecorrect insert size were sequenced using BigDye 3.1 andthe vector forward and reverse primers, and an internalprimer (525F: 5'-AAGTCTGGTGCCAGCAGCC-3') withan Applied Biosystems 3730S 48-capillary sequencer.The above processes was repeated on one additional cellof Auranticordis quadriverberis that were sampled and iso-lated at different times, in order to assure authenticity ofthe obtained sequences. Complete sequences of the SSUrDNA from the two different isolates were deposited intoGenBank [GenBank:EU484393 and Gen-Bank:EU484394].Sequence alignment and phylogenetic analysesSequences were assembled and edited using Sequencher™(version 4.5, Gene Codes Corporation, Ann Arbor, Mich-igan, USA). Acquired sequences were initially identifiedby BLAST analysis. New SSU rDNA sequences derivedfrom two different isolated of Auranticordis quadriverberiswere aligned with ClustalW [64] using the MEGA (Molec-ular Evolutionary Genetics Analysis) program version 4[65] and further refined by eye using MacClade [66].Three multiple sequence alignments were created: (1) a69-taxon global alignment comprising sequences of repre-sentatives from all major eukaryotic groups (1,134 unam-biguous sites: data not shown); (2) a 126-taxon cercozoanalignment consisting of cercozoan representatives andextensive environmental sequences (981 unambiguoussites: data not shown); and (3) a 32-taxon cercozoanalignment excluding the shorter and unrelated environ-mental sequences (1,526 unambiguous sites). All gapswere excluded from the alignments prior to phylogeneticanalyses. The alignment files are available upon request.MrBayes version 3.1.2 was used to perform Bayesian anal-yses on all three datasets [67,68]. Two parallel runs werecarried out on 2,000,000 generations with the fourMarkov Chain Monte Carlo (MCMC) chains – 1 coldchain and 3 heated chains – and sampling every 50th gen-eration (tree). The first 2,000 trees in each run were dis-carded as burn-in. Branch lengths of the trees were saved.Maximum likelihood analyses were performed on allthree datasets using PhyML [69]. Input trees for each data-set were generated by BIONJ with optimisation of topol-ogy, branch lengths, and rate parameters selected. TheGeneral Time Reversible (GTR) model of nucleotide sub-stitution was chosen. The proportion of variable rates andPage 13 of 16(page number not for citation purposes)and extension at 72 °C for 105 sec; and hold at 72 °C for10 min. PCR products corresponding to the expected sizegamma distribution parameter were estimated from theoriginal dataset. Eight categories of substitution rates wereBMC Microbiology 2008, 8:123 http://www.biomedcentral.com/1471-2180/8/123selected. PhyML bootstrap trees with 100 bootstrap data-sets were constructed using the same parameters as theindividual ML trees.Sequence availabilityThe SSU rDNA nucleotide sequences included in 32-taxonanalyses for this paper are available from the GenBankdatabase under the following accession numbers: Allasdiplophysa [GenBank:AF411262], Auranticordis quadriver-beris [GenBank:EU484393 and GenBank:EU484394],Bodomorpha minima [GenBank:AF411276], Bodomorphasp. [GenBank:DQ211596], Cercomonas plasmodialis [Gen-Bank:AF411268], Cryothecomonas aestivalis [Gen-Bank:AF290539], Dimorpha-like sp.[GenBank:AF411283], Ebria tripartita [Gen-Bank:DQ303922], Euglypha rotunda [Gen-Bank:AJ418784], Exuviaella pusilla[GenBank:DQ388459], Gymnophrys cometa [Gen-Bank:AF411284], Heteromita globosa [GenBank:U42447],Lecythium sp. [GenBank:AJ514867], Massisteria marina[GenBank:AF174372], Metopion-like sp. [Gen-Bank:AF411278], Paulinella chromatophora [Gen-Bank:X81811], Proleptomonas faecicola[GenBank:AF411275], Protaspis grandis [Gen-Bank:DQ303924], Pseudodifflugia cf. gracilis [Gen-Bank:AJ418794], Pseudopirsonia mucosa[GenBank:AJ561116], Rigidomastix-like sp. [Gen-Bank:AF411279], Spongomonas minima [Gen-Bank:AF411280], Thaumatomastix sp.[GenBank:AF411261], thaumatomonadida environmen-tal sample [GenBank:EF023494], Thaumatomonas colo-niensis [GenBank:DQ211591], Thaumatomonas seravini[GenBank:AF411259], uncultured eukaryote [Gen-Bank:AB252750], uncultured eukaryote [Gen-Bank:AB252755], uncultured eukaryote[GenBank:AB252756], uncultured eukaryote [Gen-Bank:AB275058], and uncultured marine eukaryote [Gen-Bank:DQ369017].Authors' contributionsCC and BSL conceived and designed the experiments. CC,HJE, and BSL performed microscopical studies. CC con-ducted the molecular studies, the sequence alignments,and phylogenetic analyses. CC and BSL analyzed the data,drafted the manuscript, and wrote the paper. All authorshave read and approved the final manuscript.AcknowledgementsWe are grateful to Drs. T. Cavalier-Smith for helpful discussions and to A. P. Myl'nikov for providing some difficult to access literature. We thank S. A. Breglia for helping in sample collection and K. Tangthongchaiwiriya for illustrating Auranticordis quadriverberis (Figure 6). We also thank the BioIm-aging Facility at University of British Columbia for technical help on elec-tron microscopy. C. Chantangsi was supported by a national scholarship the National Science and Engineering Research Council of Canada (NSERC 283091-04) and the Canadian Institute for Advanced Research, Program in Integrated Microbial Biodiversity.References1. Fenchel T: Ecology of Protozoa: the Biology of Free-livingPhagotrophic Protists.  Berlin: Springer-Verlag; 1987. 2. 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