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Ultrastructure and molecular phylogenetic position of a novel euglenozoan with extrusive episymbiotic… Breglia, Susana A; Yubuki, Naoji; Hoppenrath, Mona; Leander, Brian S May 19, 2010

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Breglia et al. BMC Microbiology 2010, 10:145http://www.biomedcentral.com/1471-2180/10/145Open AccessR E S E A R C H  A R T I C L EResearch articleUltrastructure and molecular phylogenetic position of a novel euglenozoan with extrusive episymbiotic bacteria: Bihospites bacati n. gen. et sp. (Symbiontida)Susana A Breglia1, Naoji Yubuki1, Mona Hoppenrath1,2 and Brian S Leander*1AbstractBackground: Poorly understood but highly diverse microbial communities exist within anoxic and oxygen-depleted marine sediments. These communities often harbour single-celled eukaryotes that form symbiotic associations with different prokaryotes. During low tides in South-western British Columbia, Canada, vast areas of marine sand become exposed, forming tidal pools. Oxygen-depleted sediments within these pools are distinctively black at only 2-3 cm depth; these layers contain a rich variety of microorganisms, many of which are undescribed. We discovered and characterized a novel (uncultivated) lineage of heterotrophic euglenozoan within these environments using light microscopy, scanning and transmission electron microscopy, serial sectioning and ultrastructural reconstruction, and molecular phylogenetic analyses of small subunit rDNA sequences.Results: Bihospites bacati n. gen. et sp. is a biflagellated microbial eukaryote that lives within low-oxygen intertidal sands and dies within a few hours of exposure to atmospheric oxygen. The cells are enveloped by two different prokaryotic episymbionts: (1) rod-shaped bacteria and (2) longitudinal strings of spherical bacteria, capable of ejecting an internal, tightly wound thread. Ultrastructural data showed that B. bacati possesses all of the euglenozoan synapomorphies. Moreover, phylogenetic analyses of SSU rDNA sequences demonstrated that B. bacati groups strongly with the Symbiontida: a newly established subclade within the Euglenozoa that includes Calkinsia aureus and other unidentified organisms living in low-oxygen sediments. B. bacati also possessed novel features, such as a compact C-shaped rod apparatus encircling the nucleus, a cytostomal funnel and a distinctive cell surface organization reminiscent of the pellicle strips in phagotrophic euglenids.Conclusions: We characterized the ultrastructure and molecular phylogenetic position of B. bacati n. gen. et sp. Molecular phylogenetic analyses demonstrated that this species belongs to the Euglenozoa and currently branches as the earliest diverging member of the Symbiontida. This is concordant with ultrastructural features of B. bacati that are intermediate between C. aureus and phagotrophic euglenids, indicating that the most recent ancestor of the Symbiontida descended from phagotrophic euglenids. Additionally, the extrusive episymbionts in B. bacati are strikingly similar to so-called "epixenosomes", prokaryotes previously described in a ciliate species and identified as members of the Verrucomicrobia. These parallel symbioses increase the comparative context for understanding the origin(s) of extrusive organelles in eukaryotes and underscores how little we know about the symbiotic communities of marine benthic environments.* Correspondence: bleander@interchange.ubc.ca1 Canadian Institute for Advanced Research, Program in Integrated Microbial Biodiversity, Departments of Botany and Zoology, University of British BioMed Central© 2010 Breglia et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.Columbia, 6270 University Boulevard, Vancouver, BC V6T 1Z4, CanadaFull list of author information is available at the end of the articleBreglia et al. BMC Microbiology 2010, 10:145http://www.biomedcentral.com/1471-2180/10/145Page 2 of 21BackgroundThe Euglenozoa is a diverse group of single-celledeukaryotes consisting of three main subgroups:euglenids, kinetoplastids and diplonemids. Euglenids areunited by the presence of a distinctive pellicle, a superfi-cial system formed by four major components: theplasma membrane, a pattern of repeating proteinaceousstrips that run along the length of the cell, subtendingmicrotubules and tubular cisternae of endoplasmic retic-ulum [1]. The group is widely known for its photosyn-thetic members (e.g. Euglena and Phacus), but themajority of the species are heterotrophic (osmotrophs orphagotrophs). Photosynthetic euglenids evolved fromphagotrophic ancestors with a complex feeding apparatusand a large number of pellicle strips that facilitate a char-acteristic peristaltic cell movement called "euglenoidmovement". This combination of characters allows phag-otrophic euglenids to engulf large prey cells, such aseukaryotic algae, which eventually led to the acquisitionof chloroplasts via secondary endosymbiosis [2,3].Euglenids are closely related to kinetoplastids anddiplonemids. Kinetoplastids (a group that includes free-living bodonids and parasitic species such as Trypano-soma and Leishmania) are united by the presence of amitochondrial inclusion of distinctively arranged DNAmolecules, called a kinetoplast or kDNA [4]. Kinetoplas-tids and euglenids share several morphological features,such as flagella with hairs and heteromorphic paraxialrods (e.g. a proteinaceous scaffolding adjacent to theusual 9+2 axoneme) and mitochondria with paddle-shaped (discoidal) cristae [5-7]. Diplonemids, on theother hand, possess a large mitochondrion with flattenedcristae and apparently lack flagellar hairs [8]. The mono-phyly of the Euglenozoa has been established on the basisof both molecular phylogenetic analyses and the follow-ing morphological synapomorphies: a tripartite flagellarroot system, presence of heteromorphic paraxial rods andtubular extrusomes.Environmental sequencing of oxygen depleted sedi-ments around the world has shown that these habitatsharbour a vast and unknown diversity of microbial lin-eages [9-14]. Phylogenetic analyses of these data havehelped demonstrate the existence of several novel lin-eages associated with many different eukaryotic super-groups. Although these types of analyses are veryeffective in revealing the actual diversity of microbes liv-ing in a particular environment, these approaches alsogenerate vast amounts of "orphan" data that cannot belinked directly to organisms known from comparativemorphology. Nonetheless, some of the environmentalsequences recovered from oxygen depleted environmentsOther studies have explored and characterized themicrobial diversity in oxygen-depleted environmentsusing microscopical approaches [15-20]. This researchhas shown that a reoccurring feature of euglenozoans liv-ing in low oxygen environments is the presence of epi-symbiotic bacteria on the cell surface. Here, we report ona highly unusual (uncultivated) euglenozoan isolatedfrom oxygen depleted marine sediments that is coveredwith two very different morphotypes of episymbionts.We characterized this lineage with light microscopy,SEM, comprehensive TEM, and molecular phylogeneticanalyses of SSU rDNA sequences. Our data demonstratethat this organism is the earliest diverging member of theSymbiontida, which is an emerging subclade of eugleno-zoans composed of anaerobic and microaerophilic flagel-lates with a superficial layer of mitochondrion-derivedorganelles that associates closely with a uniform layer ofepisymbiotic bacteria [19]. Moreover, the comparativeultrastructural data from this novel lineage sheds consid-erable light onto the phylogenetic position of the Symbi-ontida, as a whole, within the Euglenozoa.ResultsGeneral MorphologyThe cells of Bihospites bacati n. gen. et sp. were elongatedwith a somewhat rounded posterior end and were 40-120μm long and 15-30 μm wide (n = 200). The cells con-tained a brownish (or greenish) body near the posteriorend of the cell and a variable number of distinctive blackbodies at the anterior half of the cell (Figure 1A, B). Thecells of B. bacati had two heterodynamic flagella thatwere inserted subapically within a depression. The longeranterior (dorsal) flagellum extended forward and contin-uously probed the substrate during 'gliding' movements(Figure 1B); periodically, the tip of the anterior flagellumwould adhere to the substrate and abruptly drag the cellforward. The recurrent (posterior) flagellum was slightlylonger than the cell body and trailed freely beneath thecell. The cells of B. bacati were plastic and capable ofrhythmic deformations ranging from contracted torelaxed states that were reminiscent of "euglenoid move-ment" (Figure 1C). The cells divided from anterior to pos-terior along the longitudinal axis (Figure 1D). Cystformation or sexual reproduction was not observed. Cellsof B. bacati were found all year round, although the abun-dance of this species decreased significantly during thewinter months.Cell SurfaceThe cell surface of B. bacati was covered with two differ-ent morphotypes of episymbiotic bacteria: (1) morecluster with euglenozoans in phylogenetic analyses butwith no clear position within the group [9-11].abundant rod shaped episymbionts and (2) spherical-shaped episymbionts (Figure 1E, 2). The rod-shaped epi-symbionts were 3-5 μm long and were arranged in bands,Breglia et al. BMC Microbiology 2010, 10:145http://www.biomedcentral.com/1471-2180/10/145Page 3 of 21Figure 1 Light micrographs (LM) of living cells of Bihospites bacati n. gen. et sp. A. LM showing distinctive black bodies (white arrow) and the prominent nucleus (N) positioned near the anterior end of the cell. B. LM showing the extended dorsal flagellum (Df) that is inserted subapically. C. LM showing the dorsal flagellum (Df) and a contracted cell with raised helically arranged striations (S) on the surface. D. LM showing a cell dividing along the anteroposterior axis. E. LM showing rows of spherical-shaped bacterial episymbionts on the cell surface (arrowheads). F. LM showing the nucleus with a distinct thickening (arrow), providing evidence for the shape and orientation of the C-shaped rod apparatus.Breglia et al. BMC Microbiology 2010, 10:145http://www.biomedcentral.com/1471-2180/10/145Page 4 of 21Figure 2 Scanning electron micrographs (SEM) of Bihospites bacati n. gen. et sp. A. Ventral view of B. bacati showing a cell covered with rod-shaped and spherical-shaped episymbiotic bacteria (white arrowheads and black arrowheads, respectively), the vestibulum (vt), dorsal flagellum (Df) and ventral flagellum (Vf) (bar = 15 μm). B. High magnification of the vestibular opening (vt), showing the open cytostome (white arrowhead), and the dorsal (Df) and ventral flagella (Vf) without flagellar hairs. C. High magnification SEM showing the posterior end of B. bacati, in ventral view, and the external appearance of the raised articulation zones between S-shaped folds in the host cell surface (black arrowheads). The white arrows show pores on the cell surface. D. High magnification SEM showing the rod-shaped (white arrowheads) and spherical-shaped episymbionts. E. High mag-nification SEM of the spherical-shaped episymbionts showing discharged threads (black arrows) through an apical pore (bar = 0.5 μm). The white ar-row shows the initial stages of the ejection process. (B-D bar = 1 μm).Breglia et al. BMC Microbiology 2010, 10:145http://www.biomedcentral.com/1471-2180/10/145Page 5 of 21about 7 μm wide, along the longitudinal axis of the hostcell (Figure 2A). These bands peeled off when the hostcell deteriorated. The longitudinal bands of rod-shapedepisymbionts were separated and defined by single ordouble rows of spherical episymbionts, each about 0.6μm in diameter (Figure 2A-E). These longitudinal rowsusually extended nearly the entire length of the host celland were helically organized when the host cells were in acontracted state (Figure 1C, 2A). The rod-shaped episym-bionts were connected to the plasma membrane of thehost by a glycocalyx-like material (Figure 3A-E). Thespherical-shaped episymbionts were attached to the hostwithin a corresponding concavity in the host plasmamembrane (Figure 3E). The spherical-shaped episymbi-onts were highly organized and possessed an extrusiveapparatus consisting of an apical "operculum" and atightly coiled internal thread around a densely stainedcore (Figure 3D-F). The coiled thread was capable ofrapid discharge through an apical pore when disturbedduring chemical fixation for electron microscopy (Figure2A, D-E); the densely stained core was discharged first,and the coiled thread followed (Figure 3F).The ultrastructure of the host cell surface, beneath theepisymbionts, consisted of a plasma membrane that wasorganized into a repeated series of S-shaped folds (i.e.,"strips") (Figure 1C, 3A), a thin layer of glycoprotein, anda corset of microtubules (Figure 3C). The longitudinalrows of spherical-shaped episymbionts were associatedwith the troughs of the S-shaped folds (Figure 3A). Theraised articulation zones between the S-shaped foldswere visible in (i) light micrographs of contracted cells(Figure 1C), (ii) scanning electron micrographs near theposterior end of the host cell (Figure 2C), and (iii) trans-mission electron micrographs (Figure 3A). The corset ofmicrotubules beneath the folds formed a continuous rowand was linked together by short "arms" (Figure 3C).Tubular cisternae of endoplasmic reticulum and a layer ofdouble-membrane bound mitochondrion-derived organ-elles (MtD) were positioned immediately below thesuperficial corset of microtubules (Figure 3A-C, E-F). Themitochondrion-derived organelles contained a granularmatrix and none or very few cristae per TEM profile (Fig-ure 3B). There was no evidence of kinetoplast-like inclu-sions or any other kind of packed DNA within the matrixof the mitochondrion-derived organelles.The cytoplasm of the host cell was highly vacuolatedand contained clusters of intracellular bacteria withinvacuoles (Figure 4A). Batteries of tubular extrusomes,ranging from only a few to several dozen, were also pres-ent within the host cytoplasm (Figure 4B). The extru-somes were circular in cross-section and had a denselyapproximately 4 μm long, and many of them were posi-tioned immediately beneath the raised articulation zonesbetween the S-shaped surface folds (Figure 3A, 4D).Nucleus, C-shaped Rod Apparatus, Cytostomal Funnel and VestibulumThe nucleus of B. bacati was positioned in the anteriorhalf of the cell and had permanently condensed chromo-somes (Figure 1A, 5A). The nucleus was also closelylinked to a robust rod apparatus (Figure 1F). Serial sec-tions through the entire nucleus demonstrated that a C-shaped system of rods formed a nearly complete ringaround an indented nucleus (Figure 5A, 6, 7, 8 and 9). TheC-shaped system of rods consisted of two main elements:(1) a main rod that was nestled against the indentednucleus (Figure 7, 8 and 9) and (2) a folded accessory rodthat was pressed tightly against the outer side of the mainrod for most of its length. We refer to this two-partedarrangement as the "C-shaped rod apparatus" (Figure 5A,6, 7, 8 and 9). The main rod was composed of a densecluster of parallel lamellae that often appeared corru-gated, while the accessory rod was composed of striatedfibres (SF) (Figure 5A-B, 6, 7 and 8). Granular bodies ofapproximately 35 nm in diameter were observed in thespaces between the parallel lamellae of the main rod (Fig-ure 5B). The ventral side of the main rod was embeddedin an amorphous matrix that became thinner toward theposterior end of the cell, until it disappeared altogether(Figure 6A-D). A single row of longitudinal microtubuleslined the external side of the main rod, which delimitedthe boundary between the main rod and the accessoryrod for most of their length (Figure 5A-B).The anterior ends of both C-shaped rods terminatednear the antero-ventral region of the nucleus (Figure 9).The posterior end of the main rod was positioned withinthe posterior region of a feeding pocket (Figure 5C-F, 9).This feeding pocket merged together with the flagellarpocket and formed a common subapical concavity in thecell or a "vestibulum" (Figure 2B, 5, 9A). A novel "cytos-tomal funnel" was positioned at the junction, and there-fore demarcated the boundary, between the feedingpocket and the flagellar pocket (Figure 5, 6, 9A). Thecytostomal funnel was an anterior extension of the poste-rior end of the accessory rod that eventually openedwithin the subapical vestibulum (Figure 2B, 5, 6 and 9A).Some microtubules associated with the posterior end ofthe accessory rod also extended toward the ventral side ofthe cell and appeared to become continuous with the(ventral flagellar root) microtubules that reinforced theflagellar pocket (not shown).The posterior region of the feeding pocket also con-stained outer region that surrounded a lighter, granularcore; a cruciform element was observed in cross-sectionof some extrusomes (Figure 4C). The extrusomes weretained a "congregated globular structure" (CGS) that wasassociated with the posterior end of the main rod (Figure6A-B). The posterior end of the folded accessory rodBreglia et al. BMC Microbiology 2010, 10:145http://www.biomedcentral.com/1471-2180/10/145Page 6 of 21Figure 3 Transmission electron micrographs (TEM) of the cell surface of Bihospites bacati n. gen. et sp. A. Cross-section of cell showing a series of S-shaped folds in the cell surface. Elongated extrusomes (E) positioned beneath the raised articulation zones between the S-shaped folds (S). Cell surface covered with rod-shaped bacteria (black arrowheads), in cross section, and spherical-shaped bacteria (white arrowheads). Mitochondrion-de-rived organelles (MtD) underlie the cell surface. (bar = 1 μm). B. TEM showing mitochondrion-derived organelles (MtD) with zero to two cristae (arrow). Arrowheads show transverse profiles of rod-shaped episymbionts on cell surface. C. High magnification TEM of the host cell surface showing glycog-alyx (GL) connecting episymbionts to plasma membrane. Plasma membrane subtended by a thick layer of glycoprotein (double arrowhead) and a continuous row of microtubules linked by short 'arms' (arrowhead). Mitochondrion-derived organelles (MtD) positioned between the row of micro-tubules and the endoplasmic reticulum (ER). D. Oblique TEM section of spherical-shaped episymbiont showing electron-dense apical operculum (black arrow) and the extrusive thread coiled around a densely stained core region (white arrow). E. High magnification TEM of cell surface showing mitochondrion-derived organelles (MtD), rod-shaped episymbionts (arrowheads), and spherical-shaped episymbiont (black arrow) sitting within a corresponding concavity in the host cell. Core region of the spherical-shaped episymbiont (white arrow) in longitudinal section. F. TEM of spherical-shaped episymbiont showing discharged extrusive thread (arrow). Electron-dense material corresponding to the core is positioned at the tip of the discharged thread (arrow). Arrowheads indicate rod-shaped bacteria on cell surface (B-F bar = 500 nm).Breglia et al. BMC Microbiology 2010, 10:145http://www.biomedcentral.com/1471-2180/10/145Page 7 of 21became more robust as the serial sections moved fromthe posterior end of the feeding pocket toward the poste-rior end of the cell (Figure 6, 9). The posterior end of thefolded accessory rod was initially positioned between thefeeding apparatus and the flagellar apparatus; the acces-sory rod then gradually became more robust and moretightly associated with the main rod as both of the rodsmigrated around the posterior side of the nucleus andtoward the dorsal side of the nucleus (Figure 6A-D, 9).Moreover, as the sections continued posteriorly, the feed-ing pocket and the CGS that surrounded the main roddiminished, and ultimately only the main rod and theaccessory rod remained (Figure 6C-D).Serial sections through the anterior region of thenucleus, moving from anterior to posterior, demon-strated the C-shaped curvature of the rod apparatus (Fig-ure 7, 9). These sections also demonstrated how theanterior ends of both the main rod and the accessory rodterminate on the ventral side of the indented nucleus nearthe vestibulum (Figure 7F). Similarly, serial sectionsFlagellar Root SystemTwo flagella emerged from the base of the flagellar pocket(Figure 2A-B, 10A-F, 11A-E). Each flagellum had a parax-ial rod (PR) in addition to the 9+2 arrangement of micro-tubules forming the axoneme (Figure 10G-H, 11F). ThePR in the dorsal flagellum (Df) had a whorled disposition,whereas the PR of the ventral flagellum (Vf) had a lattice-like arrangement of parallel fibres (Figure 11F). No mas-tigonemes were observed on either flagellum (Figure 2A-B). The dorsal basal body contained a long opaque core(Figure 11B). Both basal bodies were approximately 1.7μm long and were linked by a connecting fibre (CF) (Fig-ure 10A-B). A cartwheel structure was present at theproximal end of both basal bodies (Figure 10A-B). Twoaccessory basal bodies (Db' and Vb') were observed onthe ventral side of the Db and the dorsal side of the Vb(Figure 10B).The flagellar root system is described here from theproximal to the distal end of the basal bodies as viewedfrom the anterior end of the cell. The basal bodies wereFigure 4 Transmission electron micrographs (TEM) of Bihospites bacati n. gen. et sp. showing intracellular bacteria and extrusomes. A. TEM showing a cell containing numerous intracellular bacteria (arrowheads) within vacuoles. B. Transverse TEM showing a battery of extrusomes (arrows) (A, B, bar = 500 nm). C. High magnification TEM of extrusomes showing a dense outer region (arrowhead) and a granular core containing a lighter cruciform structure (white arrow). Black arrow denotes the plasma membrane of the host (bar = 100 nm). D. TEM showing a longitudinal section of an extrusome; the proximal end is indicated with a black arrow. Arrowheads denote rod-shaped bacteria on the cell surface (bar = 500 nm).through the posterior region of the nucleus, moving fromanterior to posterior, demonstrated the C-shaped curva-ture of the rod apparatus and its relationship to theindented nucleus (Figure 8, 9).associated with three asymmetrically arranged flagellarroots. A dorsal root (DR) originated from the dorsal-rightside of the Db (Figure 10B, 11B-C) and was formed ofapproximately six microtubules (Figure 10E). A ventralBreglia et al. BMC Microbiology 2010, 10:145http://www.biomedcentral.com/1471-2180/10/145Page 8 of 21Figure 5 Transmission electron micrographs (TEM) of non-consecutive serial sections of Bihospites bacati n. gen. et sp. through the vestib-ular region of the cell. A. TEM showing the nucleus (N) with condensed chromatin, the dorsal side of the C-shaped rod apparatus consisting of the main rod (r) and the accessory rod (ar), and the vestibulum (vt). Several rod-shaped bacteria (black arrows) and spherical-shaped bacteria line inner surface of the vestibulum (vt) (bar = 10 μm). B. High magnification view of the C-shaped rod apparatus in Figure A showing the single row of micro-tubules (arrowheads) positioned at the junction between the tightly connected rod and accessory rod. Granular bodies (arrows) are present between the parallel lamellae that form the main rod (bar = 500 nm). C, D. Transverse TEMs showing the cytostomal funnel (cyt) and two separate lobes of the feeding pocket (arrowheads). Bacterial profiles can be seen inside the feeding pocket (arrows). Figure D uses color to distinguish between the feeding pocket (red), the vestibulum (blue), and the two branches of the flagellar pocket (green). E, F. Transverse TEMs at a more posterior level than in Figure C-D showing the posterior end of the main C-shaped rod (arrow) emerging within the posterior end of the feeding pocket. The cytostomal funnel (arrowheads) opens and fuses with the feeding pocket. Figure F uses color to distinguish between the feeding pocket (red), the vestibulum (blue), and the two branches of the flagellar pocket (green). (C-F bar = 2 μm).Breglia et al. BMC Microbiology 2010, 10:145http://www.biomedcentral.com/1471-2180/10/145Page 9 of 21Figure 6 Transmission electron micrographs (TEM) of non-consecutive serial sections through the flagellar apparatus and feeding pockets of Bihospites bacati n. gen. et sp. TEMs taken at levels posterior to those shown in Figure 5 and presented from anterior (A) to posterior (D). A. TEM showing the posterior end of the main C-shaped rod (r) embedded in an amorphous matrix (double arrowhead) and surrounded by a thick membrane with fuzzy material (arrowhead). At this level, the rod is associated with 'congregated globular structure' (CGS), and the striated fibres that form the accessory rod (ar) appear near the cytostomal funnel (cyt) at the junction between the feeding pocket and the flagellar pocket. Inset: TEM showing the accessory rod (ar) in a subsequent posterior section, as it starts to open up. Vf = ventral flagellum; Df = dorsal lagellum. B. TEM showing the sepa-ration (arrowhead) of the feeding pocket (asterisks) from the flagellar pocket (FP) near cytostomal funnel (cyt) and the expanding accessory rod (ar). C. TEM showing the diminishing feeding pocket (asterisks), the cytostomal funnel (cyt), and the separate flagellar pocket (FP). D. TEM showing the accessory rod (ar) with its characteristically folded shape becoming more tightly linked to the main rod (r). Lobes of the feeding pocket (asterisk) and the flagellar pocket (FP) are also still visible. MtD = mitochondrion-derived organelle; double arrowheads = spherical-shaped episymbionts. (bars = 2 μm).Breglia et al. BMC Microbiology 2010, 10:145http://www.biomedcentral.com/1471-2180/10/145Page 10 of 21root (VR) connected to the dorsal-right side of the ventralbasal body (Figure 11A, D-E) and was comprised initiallyof four microtubules (Figure 10D). An intermediate root(IR), originally formed of about eight microtubules (Fig-ure 10F), emerged from the left side of the Vb (Figure10C-D). The ventral root and the intermediate roots ulti-mately fused, forming a continuous VR-IR row of micro-tubules around the flagellar pocket (Figure 10G-H). Aband of dorsal microtubules (DMt), not directly associ-ated to the basal bodies, lined the dorsal side of the flagel-lar pocket (Figure 10C, F; 11A-E). Toward the anteriorend of the cell, the number of microtubules increased oneby one, until the band reached the dorsal root (DR). TheDMt and the DR eventually fused and formed a singleband of microtubules around the flagellar pocket (Figureand a ventral lamina (VL), respectively (Figure 10C-H).Both laminae extended anteriorly and ended up reinforc-ing the walls of the flagellar pocket (Figure 10G-H). TheDR, together with the DL, supported the dorsal-left sideof the pocket, and the DMt supported the dorsal-rightside. The VR - reinforced by the VL - lined the ventralside of the pocket and was in contact with the IR thatlined the ventral-left side of the flagellar pocket. Themicrotubules of the DMt and the VR became part of theelements forming the cytostomal funnel and accessoryrod (i.e., the C-shape rod apparatus in general), and boththe DR and the IR became part of the sheet of microtu-bules underlining the plasma membrane of the entire cell.Molecular Phylogenetic PositionFigure 7 Transmission electron micrographs (TEM) of non-consecutive serial sections through the anterior part of the nucleus of Bihospites bacati n. gen. et sp. Figures 7A-F are presented from anterior to posterior. A. TEM showing the nucleus (N) and the accessory rod (ar) surrounded by electron-dense material (Images are viewed from the anterior side of the cell: D, dorsal; L, left side of the cell; R, right side of the cell; V, ventral). B-C. TEMs showing the main rod (r) near the striated fibres (SF) of the accessory rod (arrow). D. TEM showing the left side of the nucleus (N) appearing behind the rod (r) and accessory rod (ar). The white arrow shows the presence of bacteria near the rod. E. TEMs showing the main rod (r) and the accessory rod (arrowheads) on the dorsal and ventral sides of the nucleus. F. Transverse TEM at the level of the vestibulum showing the disappearance of the ventral side of the main rod (r) and the drastic reduction of the accessory rod (arrowhead). Note the indentations in the nucleus for accommo-dating the main rod and accessory rod (A bar = 500 nm; B-F bar = 2 μm).10G-H).The DR and VR were associated with two electrondense bodies that elongated to form a dorsal lamina (DL)In order to infer the phylogenetic position of B. bacati, wePCR-amplified and sequenced the nearly complete SSUrDNA gene (2057 bp) from two independent isolates. TheBreglia et al. BMC Microbiology 2010, 10:145http://www.biomedcentral.com/1471-2180/10/145Page 11 of 21sequences contained expansions typical of euglenozoanSSU rDNA genes. First, we carried out a 40-taxon Maxi-mum likelihood (ML) analysis that included sequencesincluded 37 taxa representing all of the major lineages ofeuglenozoans. The phylogenetic analyses showed that theeuglenozoan sequences clustered in five main subgroupsFigure 8 Transmission electron micrographs (TEM) of non-consecutive serial sections through the posterior part of the nucleus of Bihos-pites bacati n. gen. et sp. Figures 8A-D are presented from anterior to posterior. A-C. TEMs showing the rod (r) and the folded accessory rod (ar) nes-tled within indentations in the dorsal and ventral sides of the nucleus. The ventral part of the accessory rod runs close to the main rod for most of its length and extends toward the flagella on the ventral side of the cell. N = nucleus; D, dorsal; L, left side of the cell; R, right side of the cell; V, ventral; Images are viewed from the anterior side of the cell. D. TEMs showing the main rod (r) and the accessory rod (ar) reaching the posterior end of the nucleus (N). The main rod consists of parallel-arranged lamellae. Most of the nucleus and the main rod have disappeared from the section. The acces-sory rod (ar) consists of striated fibres that wrap around the main rod and the nucleus (bars = 2 μm).representing all of the major groups of eukaryotes; theresulting phylogeny showed B. bacati grouped stronglywithin the Euglenozoa (not shown). A second analysiswith high statistical support (Figure 12): (i) a kinetoplas-tid clade, (ii) a diplonemid clade, (iii) a bacteriovorouseuglenid clade, (iv) a eukaryovorous + phototrophicBreglia et al. BMC Microbiology 2010, 10:145http://www.biomedcentral.com/1471-2180/10/145Page 12 of 21euglenid clade and (v) the Symbiontida, a newly namedclade that includes Calkinsia aureus and several environ-mental sequences. Bihospites bacati clustered with theSymbiontida with extremely high statistical support (MLbootstrap value = 100% and Bayesian posterior probabil-ity > 0.95), as the sister lineage to the rest of this group.Calkinsia aureus branched next within the Symbiontidaand formed the sister lineage to several environmentalsequences (Figure 12). However, the relationship of theSymbiontida to the other main subgroups within theEuglenozoa was unclear.DiscussionBihospites bacati n. gen et sp. possesses all three synapo-extrusomes. Concordantly, our analyses of SSU rDNAsequences clearly places B. bacati within the Euglenozoa,specifically within the Symbiontida. Several studies basedon environmental sequences indicated the existence of anovel rDNA clade of euglenozoans [9-11]. However, theSymbiontida was proposed after the ultrastructuraldescription and molecular phylogeny of C. aureusstrongly grouped this species with these environmentalsequences, as a distinct subgroup within the Euglenozoa[19]. Nonetheless, it was not clear in that study whetherthe Symbiontida was a new clade of euglenozoans or asubclade within one of the three previously recognizedmembers of the Euglenozoa (i.e., kinetoplastids, diplone-mids and euglenids). Our comprehensive characteriza-Figure 9 Diagrams showing a reconstruction of the ultrastructure of Bihospites bacati n. gen. et sp. Relationships between C-shaped rod ap-paratus, nucleus, cytostomal funnel, feeding pocket, flagellar pocket and vestibulum, as inferred from serial transmission electron microscopy (TEM), scanning electron microscopy (SEM), and light microscopy (LM). A. Cell viewed from the right side showing the positions of the nucleus (N), the C-shaped main rod (r), the accessory rod (ar), and the cytostomal funnel (cyt) in relation to the feeding pocket (FeP), the flagellar pocket (FP) and the vestibulum (vt); Vf = ventral flagellum; Df = dorsal flagellum; Db = dorsal basal body; Vb = ventral basal body. B. Diagram emphasizing the relationship between nucleus (N), main rod (r), and folded accessory rod (ar). The diagram is divided into three sections; and the nucleus removed from the top section for clarity. Posterior end of the main rod positioned at the level of the vestibulum on the ventral side of the nucleus. This rod extends posteriorly and then encircles the posterior, dorsal and anterior ends of the nucleus before terminating on the ventral side of the nucleus just above the vestibu-lum; therefore, this rod is C-shaped. The folded accessory rod runs along the C-shaped main rod for most of its length, terminating at the same point just above the vestibulum; however, on the ventral side of the nucleus, the posterior end of the accessory rod extends both anteriorly, defining the cytostomal funnel (cyt), and ventrally toward the ventral basal body.morphies that unify the Euglenozoa: a tripartite flagellarroot system, heteromorphic paraxial rods and tubulartion of B. bacati sheds considerable light onto thisquestion.Breglia et al. BMC Microbiology 2010, 10:145http://www.biomedcentral.com/1471-2180/10/145Page 13 of 21Remnants of Pellicle StripsBihospites bacati possesses a cell surface consisting of S-shaped folds, microtubules and endoplasmic reticulumthat is similar to the pellicle of S-shaped strips found ineuglenids. In most photosynthetic euglenids, the pelliclestrips usually consist of a robust proteinaceous frame thatsupports and maintains the shape of the cell, even duringeuglenoid movement [21-23]. However, like in mostphagotrophic euglenids, there is no robust proteinaceousframe in B. bacati. Articulation zones between strips inthe euglenid pellicle function as 'slipping points' aroundwhich the pellicle can change shape rather freely; more-cell surface in B. bacati, it was not possible to determinean exact number of S-shaped folds in the cell surface.Nonetheless, the microtubular corset in most euglenids isregularly interrupted, thus forming groups of a fewmicrotubules associated with each pellicle strip, the num-ber of which varies between species [21-23]. By contrast,the microtubules beneath the plasma membrane in B.bacati form a continuous corset over the entire cell, muchlike that found in several phagotrophic euglenids (e.g.,Dinema [21]) and in symbiontids (C. aureus [19] andPostgaardi mariagerensis [16]).Figure 10 TEM micrographs showing sections of basal bodies, flagellar roots and associated structures, of Bihospites bacati n. gen. et sp. A-H from proximal to distal end of flagellar pocket. A-C. Non-consecutive serial sections showing origin and organization of flagellar pocket. A. High magnification TEM of proximal region of basal bodies showing dorsal and ventral basal bodies (Db and Vb) linked by a connecting fibre (CF). Basal bodies with cartwheel structures associated to electron-dense fibres (arrowheads). B. TEM showing accessory dorsal and ventral basal bodies (Db' and Vb') on the left of the two main basal bodies. Dorsal root (DR) connects to electron-dense body (dorsal lamella=DL), on right side of Db. C. TEM show-ing intermediate root (IR) associated with right side of Vb. Ventral root (VR) associated with electron-dense material that becomes ventral lamella (VL). Row of dorsal microtubules (DMt), not associated with basal bodies. D. Detail of ventral side of Figure C showing Vb, VR formed by four microtubules, VL and intermediate root (arrowhead), initially composed of eight microtubules. E. Detail of dorsal side of Figure C showing DR, with six microtubules (white arrowheads), and DL. F. TEM showing three flagellar roots and DMt around flagellar pocket. Df = dorsal flagellum; Vf = ventral flagellum. G-H. Non-consecutive serial TEM sections of flagellar pocket showing Df and Vf with paraxial rods (PR), flagellar roots, DMt of microtubules lining flagellar pocket, and DL and VL. (A-B and D-E bars = 200 nm; C and F bars = 500 nm; G-H bars = 2 μm)over, the relative number of strips in each euglenid spe-cies reflects phylogenetic relationships and the degree ofcell plasticity [24]. Due to the extreme flexibility of theA Novel Feeding Apparatus Consisting of RodsBihospites bacati possesses a well-developed C-shapedrod apparatus consisting of a main rod and an associatedBreglia et al. BMC Microbiology 2010, 10:145http://www.biomedcentral.com/1471-2180/10/145Page 14 of 21Figure 11 Transmission electron micrographs (TEM) of Bihospites bacati n. gen. et sp. showing the emergence and organization of the fla-gella. A. Longitudinal TEM through the electron-dense region near the origin of the basal bodies. The ventral root (VR) originates from the ventral basal body (Vb). A row of microtubules (DMt) lines the dorsal side of the incipient flagellar pocket. B. Longitudinal TEM through the dorsal flagellum showing the dorsal basal body (Db) associated with the dorsal flagellar root (DR), the ventral basal body (Vb), and the dorsal microtubules (DMt). C-D. TEM sections showing the dorsal flagellum (Df) and the intermediate root (IR) associated with the ventral basal body (Vb). E. TEM showing oblique sections through both flagella and the positions of the VR, IR and DMt in the flagellar pocket. The electron-dense material from which the flagellar apparatus originated in Figure A elongates to form the dorsal lamella (DL). The double arrowheads show the paraxial rod in the ventral flagellum (Vf). F. Transverse TEM of the Df and Vf showing the 9+2 arrangement of microtubules in the axoneme and the heteromorphic paraxial rods (PR). (A-E bars = 500 nm; F bar = 200 nm)Breglia et al. BMC Microbiology 2010, 10:145http://www.biomedcentral.com/1471-2180/10/145Page 15 of 21Figure 12 Phylogenetic position of Bihospites bacati n. gen. et sp. within the Euglenozoa as inferred from small subunit (SSU) rDNA se-quences. Maximum likelihood (ML) analysis of 35 euglenozoan taxa, rooted with two jakobids (Andalucia incarcerata and A. godoyi). Only ML boost-raps greater then 50% are shown. Thick branches correspond to Bayesian posterior probabilities over 0.95. Ba, bacterivorous taxa; Eu, eukaryovorous taxa; Ph, photosynthetic taxa.accessory rod. Several heterotrophic euglenids [25-30], sory rods (e.g. Peranema trichophorum has two mainand some species of diplonemids [31-36], have beendescribed with feeding apparatuses consisting of twomain rods; some species also have corresponding acces-rods and two folded accessory rods) or have a branchedrod that gives the appearance of three main rods (e.g.,Entosiphon). Nonetheless, there are several differencesBreglia et al. BMC Microbiology 2010, 10:145 Page 16 of 21http://www.biomedcentral.com/1471-2180/10/145between these rods and those described here for B.bacati. Firstly, B. bacati only has one main rod and onefolded accessory rod; this configuration has never beendescribed so far. Secondly, the vast majority of this appa-ratus tightly encircles the nucleus in a C-shaped fashion,the functional significance of which is totally unclear. Thestraight rods in euglenids support and line a conspicuousfeeding pocket, whereas the feeding pocket in B. bacationly associates with the posterior end(s) of the C-shapedrod apparatus. Thirdly, the main C-shaped rod in B.bacati is formed by a highly novel arrangement of tightlypacked lamellae, and only a single row of microtubulesoriginating from the VR separates the main C-shaped rodfrom the folded accessory rod. This row of microtubulesdemarcates the end of each lamella in the main rod. In allof the previously described euglenozoan species, differentrods are formed by different proportions of amorphousmaterial (not parallel lamellae) and microtubules origi-nating from the ventral root of the ventral basal body.Fourthly, the posterior terminus of the accessory rod in B.bacati participates in the formation of a novel cytostomalfunnel that extends anteriorly and merges with the sub-apical vestibulum. The cytostomal funnel presumablycloses the connection between the flagellar pocket andthe vestibulum during feeding. Although the cytostomalfunnel in B. bacati is likely homologous to the "vanes"described in several different phagotrophic euglenids, theunusual ultrastructural features of B. bacati made thisinference somewhat tenuous. Nonetheless, the additional"congregated globular structure" (CGS) at the posteriorend of the main rod in B. bacati is also present in Calkin-sia aureus [19]. However, the feeding apparatus in C.aureus lacks conspicuous rods (or vanes) and consistsmainly of a feeding pocket reinforced by microtubulesfrom the VR, similar to the MTR pockets of other eugle-nozoans (e.g., Petalomonas). Overall, the C-shaped rodapparatus in B. bacati appears to contain some homolo-gous subcomponents with phagotrophic euglenozoans(e.g., a main rod and a folded accessory rod), but, as high-lighted above, this apparatus is novel in most respects.The presence of a highly plastic cell surface, an elabo-rate feeding apparatus, and brownish bodies, reminiscentof food vacuoles, suggests that B. bacati is capable ofengulfing large prey cells such as other eukaryotes[1,3,24,27,29,37]; however, this species was never directlyobserved preying on (relatively large) microeukaryoticcells present in the environment. Nonetheless, the pres-ence of intracellular bacteria surrounded by vacuolesnear the feeding pocket indicates that B. bacati activelyfeeds on bacteria. It is also possible that B. bacati feeds onthe rod shaped episymbiotic bacteria that grow over theExtrusomesTubular extrusomes are present in several members ofthe Euglenozoa [16,19,36] and constitute a synapomor-phy for the group. Among the Symbiontida, C. aureus hastubular extrusomes clustered in a single large battery thatis longitudinally arranged and anchored to a novel "extru-somal pocket" [19]. Although Bihospites bacati also pos-sesses tubular extrusomes, these organelles are notorganized as a single battery. The extrusomes in B. bacatiare arranged in several smaller clusters that are distrib-uted in different places throughout the superficial cyto-plasm; solitary extrusomes are organized consecutivelybeneath the articulation zones of the S-shaped pellicularfolds or "strips". A similar arrangement of tubular extru-somes has also been observed in P. mariagerensis [16].Episymbiotic BacteriaSeveral distantly related species of euglenozoans havebeen described with episymbiotic bacteria. These eugle-nozoans are usually phagotrophs that live in oxygen-depletd to anoxic marine environments, such as that inwhich B. bacati thrives [15,16,18,19,38,39]. However, twospecies of euglenids living in well-oxygenated, freshwaterenvironments have also been described as having episym-biotic bacteria: the phototroph Euglena helicoideus [40],and the phagotroph Dylakosoma pelophilum [41]. Theepisymbionts so far encountered in euglenozoans areeither rod-shaped (in Euglena helicoideus [40], Postgaardimariagerensis [16], Calkinsia aureus [19,38]) or spheri-cal-shaped (D. pelophilum [41]). Bihospites bacati, how-ever, is the first euglenozoan described with bothmorphotypes of episymbionts.Hypotheses about the role of rod-shaped bacteria insymbiotic relationships with eukaryotic hosts usuallyemphasize commensalism, where the bacteria benefitfrom metabolic byproducts secreted by the host[15,16,20]. It has also been proposed that the rod-shapedbacteria are chemoautotrophic sulphur or methanogenic-oxydizers and form a mutualistic relationship with thehost [18], whereby the host provides anchorage for thebacteria and the bacteria detoxify the immediate environ-ment for the host [39,42]. The episymbiotic bacteria mayalso serve as a food-source for the host, as has beenobserved in one ciliate [43].Spherical episymbiotic bacteria have been reported inone other euglenozoan based only on light microscopy:the freshwater euglenid D. pelophilum [41]. However, thisspecies has so far been poorly described and morphologi-cal characteristics of the bacteria are very difficult to eval-uate; it was reported that the bacteria on the surface of D.pelophilum are 2 μm in diameter, twice the size of thosehost surface and into the subapical vestibulum. in B. bacati. Spherical episymbiotic bacteria that arenearly identical at the ultrastructural level to those weBreglia et al. BMC Microbiology 2010, 10:145http://www.biomedcentral.com/1471-2180/10/145Page 17 of 21describe here on B. bacati have been demonstrated onone species of hypotrich ciliate isolated from tidal pools[43-46]. Molecular phylogenetic evidence demonstratesthat these episymbionts, called "epixenosomes", are novellineages of verrucomicrobial bacteria, and experimentsindicate that the extrusive nature of the spherical episym-bionts function in defense against predators [43,45,46].Therefore, these episymbionts improve the comparativecontext for understanding the origin(s) of different typesof extrusive organelles in different lineages of eukaryotes(e.g., ejectosomes in cryptophytes and nematocysts incnidarians and dinoflagellates). A more comprehensiveexamination and discussion of the biology and origins ofthe epixenosomes in B. bacati have been incorporatedinto a companion paper currently in preparation for pub-lication (Breglia, Yubuki and Leander, unpubl. data).The Identity and Composition of the SymbiontidaMolecular phylogenetic analyses using SSU sequencesplace B. bacati as the earliest diverging branch within theSymbiontida. The Symbiontida are anaerobic andmicroaerophilic euglenozoans covered with rod-shapedbacteria that are in close association with a superficiallayer of mitochondrion-derived organelles with reducedor absent cristae; accordingly, it was predicted that rod-shaped episymbionts are present in most (if not all) mem-bers of the group [19]. The morphology of B. bacati isconcordant with this description, reinforcing the inter-pretation that the presence of episymbiotic bacteria is ashared derived character of the most recent ancestor ofthe Symbiontida. This hypothesis is more robustly cor-roborated when we consider that B. bacati and C. aureusform a paraphyletic assemblage near the origin of theSymbiontida. In other words, episymbiotic bacteria areno longer a character known only in a single lineagewithin this group. Given this context, current ultrastruc-tural data indicate that P. mariagerensis is also a memberof the Symbiontida (e.g., B. bacati, C. aureus and P. mar-iagerensis all lack flagellar hairs and possess rod-shapedepisymbionts, a continuous corset of cortical microtu-bules, and a superficial layer of mitochondrion-derivedorganelles) [16,19]. This inference, however, needs to beexamined more carefully with an ultrastructural charac-terization of the flagellar apparatus and feeding apparatusin P. mariagerensis and with molecular phylogenetic datafrom the host and the episymbionts.The presence of episymbiotic bacteria and the superfi-cial distribution of mitochondria with reduced cristae inB. bacati, C. aureus and P. mariagerensis indicate a mutu-alistic relationship that enabled both lineages to diversifywithin low-oxygen environments. Determining whetherreveal co-evolutionary patterns between the symbiontsand the hosts. The geographic distribution of C. aureusand B. bacati (i.e. seafloor sediments of Santa BarbaraBasin, California and coastal sediments of British Colum-bia, Canada) suggests that the Symbiontida is more wide-spread and diverse than currently known. This view issupported by the existence of related environmentalsequences originating from Venezuela, Denmark andNorway [9,11,13]. Moreover, an organism with strikingmorphological resemblance to B. bacati has been previ-ously observed in the Wadden Sea, Germany, [47]. Morecomprehensive sampling of anoxic and low-oxygen sedi-ments around the world will shed considerable light onthe abundances and ecological significance of this enig-matic group of euglenozoans.ConclusionsWe described and characterized a novel flagellate frommicro-aerobic marine sand: Bihospites bacati n. gen. etsp. Both comparative ultrastructure and molecular phylo-genetic analyses strongly support the placement of B.bacati with the Euglenozoa and, more specifically, as anew member of the Symbiontida. An early divergingposition of B. bacati within the Symbiontida is consistentwith the presence of morphological features that are tran-sitional between those found in C. aureus and phago-trophic euglenids: (1) a cell surface with strip-like S-shaped folds but lacking the proteinaceous frames of theeuglenid pellicle, (2) a compact but robust rod-basedfeeding apparatus, and (3) a dense community of rod-shaped episymbiotic bacteria on the cell surface but with-out the elaborate extracellular matrix of C. aureus. There-fore, the molecular phylogenetic position and suite ofintermediate ultrastructural features in B. bacati suggestthat the most recent ancestor of the Symbiontidadescended from phagotrophic euglenids. Although theclose association of rod-shaped episymbiotic bacteriawith the underlying mitochondria is a shared feature ofsymbiontids, the presence of extrusive verrucomicrobialepisymbionts in B. bacati is highly unusual. These rapid-firing episymbionts could provide critical context forunderstanding the origin(s) of several different types ofextrusive organelles in eukaryotes, and their discovery onthis novel euglenozoan lineage underscores how little weknow about the diverse symbiotic communities presentin marine benthic environments.MethodsCollection of organismsSediment samples were collected at low tide from theshoreline of Centennial Beach (Boundary Bay) in South-the episymbionts on B. bacati, C. aureus and other sym-biontids are closely related will more robustly establishthe identity and composition of the clade and potentiallywestern British Columbia, Canada (49° 00' 4797''N, 123°02' 1812''W), during the spring and summer of 2007 and2008. The samples were taken at a depth of 1-3 cm belowBreglia et al. BMC Microbiology 2010, 10:145http://www.biomedcentral.com/1471-2180/10/145Page 18 of 21the sediment surface, from a conspicuous layer of blacksand. The sediment samples were stored in flat containersat room temperature before individually isolated cellswere prepared for light microscopy, electron microscopyand DNA extraction. Cells were extracted from the sandsamples through a 48-μm mesh using the Uhlig meltedseawater-ice method [48].Attempts to culture the organism were made using twodifferent media: ATCC 1728 (for growing Isonema) andCCAP 1259/1 (for growing Petalomonas cantuscygni).Both media were diluted in sterile seawater and keptunder low oxygen conditions (oxygen content below 1%)using the ANAEROGEN™ COMPACT Kit system foranaerobic incubation; however, the cells did not repro-duce and disappeared within 24 hours.Light and electron microscopyDifferential interference contrast (DIC) light micrographswere taken using a Zeiss Axioplan 2 imaging microscopeand a Leica DC500 digital chilled CCD camera.Cells isolated from the British Columbia locality werefixed for scanning electron microscopy (SEM) using the4% osmium tetroxide vapour protocol described previ-ously [1]. The cells were then transferred onto a 10-μmpolycarbonate membrane filter, dehydrated with a gradedethanol series, and critical point dried with CO2 using aTousimis Critical Point Dryer. The filter was thenmounted on an aluminium stub, sputter coated withgold/palladium using a Cressington 208 HR High Resolu-tion Sputter Coater, and observed with a Hitachi S-4700field emission scanning electron microscope.Cells isolated from the surrounding sediment were pre-fixed for transmission electron microscopy (TEM) using4% (v/v) glutaraldehyde in 0.2 M sodium cacodylate buf-fer (SCB) (pH 7.2) with the addition of 0.3 M sorbitol.The pre-fixed cells were washed in 0.2 M SCB (pH 7.2)three times and embedded in 2% of low melting tempera-ture agarose and post-fixed in 1% (w/v) osmium tetroxidein 0.2 M SCB (pH 7.2) at room temperature for 1 hr,before being dehydrated through a graded series of etha-nol and 100% acetone. The dehydrated cells were theninfiltrated with acetone-Epon 812 resin mixtures and100% Epon 812 resin. Ultra-thin serial sections were col-lected on copper Formvar-coated slot grids, stained with2% (w/v) uranyl acetate and lead citrate, and observedusing a Hitachi H7600 electron microscope.DNA extraction, PCR amplification, alignment and phylogenetic analysisGenomic DNA was extracted using the MasterPureComplete DNA and RNA purification Kit (Epicentre, WI,2"). Polymerase chain reactions (PCR) were performedusing PuRe Taq Ready-To-Go PCR beads kit (GE Health-care, Buckinghamshire, UK). Nearly the entire eukaryoticSSU rDNA gene was amplified from each isolate usingthe eukaryotic universal primers 5'- TGATCCTTCTG-CAGGTTCACCTAC-3' [49] and 5'-GCGCTACCTGGT-TGATCCTGCCAGT-3' [50]. PCR amplificationsconsisted of an initial denaturing period (95°C for 3 min),35 cycles of denaturing (93°C for 45 s), annealing (5 cyclesat 45°C and 30 cycles at 55°C, for 45 s), extension (72°Cfor 2 min), and a final extension period (72°C for 5 min).The amplified DNA fragments were purified from aga-rose gels using UltraClean 15 DNA Purification Kit (MOBio, CA, USA), and subsequently cloned into the TOPOTA Cloning Kit (Invitrogen, CA, USA). Two clones of theeukaryotic SSU rRNA gene, from each of the two isolates(i.e., four clones in total), were sequenced with the ABIBig-Dye reaction mix using the vector primers and inter-nal primers oriented in both directions. The newsequences were screened with BLAST, identified bymolecular phylogenetic analysis, and deposited into Gen-Bank: HM004353, HM004354.The SSU rDNA sequences from B. bacati were ana-lyzed within the context of two alignments: (1) a 40-taxonalignment consisting of taxa representing all of the majorgroups of eukaryotes (988 unambiguously aligned sites)and (2) a 37-taxon alignment consisting of taxa repre-senting all of the major lineages of euglenozoans (760unambiguously aligned sites). Ambiguously aligned posi-tions and gaps were excluded from both analyses. Phylo-genetic relationships were inferred using maximumlikelihood (ML) and Bayesian methods with the programsPhyML [51] and MrBayes [52], respectively. For ML, thenucleotide datasets were analysed using a general-time-reversible (GTR) model of base substitutions, plus agamma correction with eight substitution rate categoriesand the proportion of invariable sites (GTR + I + G). MLbootstrap analysis of 500 replicates was performed withthe same parameters described above. For Bayesian anal-yses, the program MrBayes was set to operate with agamma correction with eight categories and proportionof invariable sites, and four Monte-Carlo-Markov chains(MCMC) (default temperature = 0.2). A total of 2,000,000generations was calculated with trees sampled every 50generations and with a prior burn-in of 100,000 genera-tions (i.e., 2,000 sampled trees were discarded). A major-ity rule consensus tree was constructed from 18,000 post-burn-in trees with PAUP* 4.0. Posterior probabilities cor-respond to the frequency at which a given node is foundin the post-burn-in trees.USA) from 30 cells that were individually isolated andwashed three times in sterile seawater (i.e., "isolate 1").This procedure was repeated three months later on a dif-ferent sample of 30 individually isolated cells (i.e., "isolateArchivingA digital archive of this paper is available from PubMedCentral and print copies are available from libraries in theBreglia et al. BMC Microbiology 2010, 10:145http://www.biomedcentral.com/1471-2180/10/145Page 19 of 21following five museums: Natural History MuseumLibrary (Cromwell Road, London, SW7 5BD, UK), Amer-ican Museum of Natural History (Department of LibraryServices, Central Park West at 79th St., New York, NY,10024, USA), Muséum national d'Histoire naturelle(Direction des bibliothèques et de la documentation, 38rue Geoffroy Saint-Hilaire, 75005 Paris, France), RussianAcademy of Sciences (Library for Natural Sciences of theRAS Znamenka str., 11, Moscow, Russia) and AcademiaSinica (Life Science Library, 128 Sec. 2 Academia Rd,Nankang Taipei 115, Taiwan R.O.C.).Formal Taxonomic DescriptionsEuglenozoa, Cavalier-Smith, 1981 [53]Symbiontida, Yubuki, Edgcomb, Bernhard & Leander,2009 [19]Bihospites n. gen. Breglia, Yubuki, Hoppenrath andLeander 2010DescriptionUninucleate biflagellates; two heterodynamic flagellainserted subapically, with paraxial rods and no mas-tigonemes; flagella of approximately the cell length; elon-gated cells with a rounded posterior end; nucleus atanterior end of cell; cell covered with epibiotic bacteria oftwo different types: rod-shaped and spherical-shaped;cell surface with S-shaped folds; tubular extrusomes withcruciform core; presence of black bodies mainly at theanterior end of cell; rhythmic cell deformations and glid-ing motility.Type speciesBihospites bacati.EtymologyLatin Bihospites, with two guests. The generic namereflects the presence of two different episymbiont mor-photypes: rod-shaped, and spherical-shaped episymbi-onts.Bihospites bacati n. sp. Breglia, Yubuki, Hoppenrathand Leander 2010DescriptionCell elongated with rounded ends; cell size 40-120 μm inlength and 15-30 μm in width; two heterodynamic fla-gella inserted subapically; anterior nucleus; cell coveredwith epibiotic bacteria of two different types: rod-shapedand spherical-shaped; cell surface with S-shaped folds;mitochondrion-derived organelles with reduced orabsent cristae; feeding apparatus with conspicuous C-shaped rod and accessory rod that encircles the indentednucleus; the rod is formed by tightly packed, parallel-gliding motility. Small subunit rRNA gene sequences[GenBank: HM004353, HM004354].HapantotypeBoth resin-embedded cells used for TEM and cells ongold sputter-coated SEM stubs have been deposited inthe Beaty Biodiversity Research Centre (Marine Inverte-brate Collection) at the University of British Columbia,Vancouver, Canada.IconotypesFigs 1A, 2A and 9A.Type localityTidal sand-flat at Centennial Beach, Vancouver, BritishColumbia, Canada (49°00' 4797''N, 123°02'1812''W).HabitatMarine sand, black layer 2-3 cm deep.EtymologySpecific epithet, Latin bacati, ornamented with pearls.The etymology for the specific epithet reflects the pres-ence of distinct longitudinal rows of spherical-shapedepisymbionts, reminiscent of pearl necklaces.Registration of new genus and species name in ZooBankLSID for article: urn:lsid:zoobank.org:pub:40211D82-B95C-494A-B8D0-7E061E80DD18LSID for the genus Bihospites: urn:lsid:zoobank.org:act:794D6C7B-BFB1-45C7-8DDA-32D44F3B0E50LSID for the species B. bacati: urn:lsid:zoobank.org:act:E1549565-5434-4F85-B936-7D0C485596B8Abbreviationsar: accessory rod; CGS: congregated globule structure; Cyt: cytostome; Db: dor-sal basal body; Db': dorsal pro-basal body; Df: dorsal flagellum; DL: dorsal lam-ina; DMt: dorsal microtubules; DR: dorsal root; E: extrusome; epi: epixenosome;ER: endoplasmic reticulum; FP: flagellar pocket; IR: intermediate root; LM: lightmicroscope; MtD: mitochondrion-derived organelle; N: nucleus; Nu: nucleolus;PR: paraxial rod; r: rod; S: strips; SF: striated fibre; SEM: scanning electron micro-scope; TEM: transmission electron microscope; tz: transition zone; Vb: ventralbasal body; Vb': ventral pro-basal-body; Vf: ventral flagellum; VL: ventral lamina;VR: ventral root; vt: vestibulum.Authors' contributionsSAB collected the sediment samples from Boundary Bay; generated the LM,SEM, and SSU rDNA sequence data; and wrote the first draft of the paper. NYgenerated the TEM data and helped with the phylogenetic analyses and inter-pretation of the TEM data. MH carried out the sampling, identification and LMwork of the German material and helped with the identification of the Cana-dian material. BSL funded and supervised the collection and interpretation ofthe ultrastructural and molecular phylogenetic data and contributed to writingthe paper. All authors have read, edited and approved the final manuscript.AcknowledgementsThis research was supported by grants from the Tula Foundation (Centre for Microbial Diversity and Evolution), National Science and Engineering Research Council of Canada (NSERC 283091-09), and the Canadian Institute for arranged lamellae; presence of black bodies, mainly at theanterior end of the cell; rhythmic cell deformations andAdvanced Research, Program in Integrated Microbial Biodiversity.Breglia et al. 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