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Ultrastructure and molecular phylogeny of Calkinsia aureus: cellular identity of a novel clade of deep-sea… Yubuki, Naoji; Edgcomb, Virginia P; Bernhard, Joan M; Leander, Brian S Jan 27, 2009

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ralssBioMed CentBMC MicrobiologyOpen AcceResearch articleUltrastructure and molecular phylogeny of Calkinsia aureus: cellular identity of a novel clade of deep-sea euglenozoans with epibiotic bacteriaNaoji Yubuki1, Virginia P Edgcomb2, Joan M Bernhard2 and Brian S Leander*1Address: 1Canadian Institute for Advanced Research, Program in Integrated Microbial Biodiversity, Departments of Botany and Zoology, University of British Columbia, 6270 University Boulevard, Vancouver, BC V6T 1Z4, Canada and 2Department of Geology and Geophysics, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USAEmail: Naoji Yubuki - yubuki@interchange.ubc.ca; Virginia P Edgcomb - vedgcomb@whoi.edu; Joan M Bernhard - jbernhard@whoi.edu; Brian S Leander* - bleander@interchange.ubc.ca* Corresponding author    AbstractBackground: The Euglenozoa is a large group of eukaryotic flagellates with diverse modes ofnutrition. The group consists of three main subclades – euglenids, kinetoplastids and diplonemids– that have been confirmed with both molecular phylogenetic analyses and a combination of sharedultrastructural characteristics. Several poorly understood lineages of putative euglenozoans live inanoxic environments, such as Calkinsia aureus, and have yet to be characterized at the molecularand ultrastructural levels. Improved understanding of these lineages is expected to shedconsiderable light onto the ultrastructure of prokaryote-eukaryote symbioses and the associatedcellular innovations found within the Euglenozoa and beyond.Results: We collected Calkinsia aureus from core samples taken from the low-oxygen seafloor ofthe Santa Barbara Basin (580 – 592 m depth), California. These biflagellates were distinctivelyorange in color and covered with a dense array of elongated epibiotic bacteria. Serial TEM sectionsthrough individually prepared cells demonstrated that C. aureus shares derived ultrastructuralfeatures with other members of the Euglenozoa (e.g. the same paraxonemal rods, microtubularroot system and extrusomes). However, C. aureus also possessed several novel ultrastructuralsystems, such as modified mitochondria (i.e. hydrogenosome-like), an "extrusomal pocket", a highlyorganized extracellular matrix beneath epibiotic bacteria and a complex flagellar transition zone.Molecular phylogenies inferred from SSU rDNA sequences demonstrated that C. aureus groupedstrongly within the Euglenozoa and with several environmental sequences taken from low-oxygensediments in various locations around the world.Conclusion: Calkinsia aureus possesses all of the synapomorphies for the Euglenozoa, but lackstraits that are specific to any of the three previously recognized euglenozoan subgroups. Molecularphylogenetic analyses of C. aureus demonstrate that this lineage is a member of a novel euglenozoansubclade consisting of uncharacterized cells living in low-oxygen environments. Our ultrastructuralPublished: 27 January 2009BMC Microbiology 2009, 9:16 doi:10.1186/1471-2180-9-16Received: 28 June 2008Accepted: 27 January 2009This article is available from: http://www.biomedcentral.com/1471-2180/9/16© 2009 Yubuki 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 22(page number not for citation purposes)description of C. aureus establishes the cellular identity of a fourth group of euglenozoans, referredto as the "Symbiontida".BMC Microbiology 2009, 9:16 http://www.biomedcentral.com/1471-2180/9/16BackgroundThe Euglenozoa is a clade of eukaryotic microorganismswith very diverse lifestyles and that tentatively falls withinone of six emerging supergroups of eukaryotes, namelythe "Excavata" [1-3]. Most euglenozoans cluster withinthree major subgroups that have been established withboth molecular phylogenetic analyses and combinationof ultrastructural characteristics (e.g. the same tripartiteflagellar root system): the Kinetoplastida, the Euglenidaand the Diplonemida [3-8]. Kinetoplastids possess mito-chondria with a uniquely structured genome, called "kine-toplast" DNA, and the group includes both free-livingphagotrophic lineages (e.g. bodonids) and parasitic line-ages (e.g. trypanosomatids such as Trypanosoma and Liesh-mania). Euglenids possess a cytoskeleton, or "pellicle",consisting of overlapping proteinaceous strips that arearranged either longitudinally or helically, and the groupincludes bacteriovorous lineages (e. g. Petalomonas),eukaryovorous lineages (e.g. Peranema), osmotrophic lin-eages (e.g. Menodinium) and photosynthetic lineages (e.g.Euglena). The mitochondria of kinetoplastids and eugle-nids possess cristae that are distinctively discoidal inshape. By contrast, diplonemids consist of only two gen-era, Diplonema and Rhynchopus, with sack-shaped cells,short flagella and flattened mitochondrial cristae andwithout kinetoplast DNA, pellicle strips, and paraxone-mal rods.Ultrastructural studies have also demonstrated lineages ofeuglenozoans that do not fall neatly within any of thethree established subgroups, such as Postgaardi mariager-ensis, which inhabits low oxygen environments and is cov-ered with epibiotic bacteria [9]. Currently, P. mariagerensisis grouped together with another poorly understoodanoxic flagellate, namely Calkinsia aureus, as incertae sediswithin the Euglenozoa [3]; although molecular data isunavailable for both species, one author has chosen toclassify them within a taxon called the "Postgaardea"[10,11]. C. aureus was originally collected from anoxicsediments near Woods Hole, MA (USA) and describedwith only line drawings as a member of the euglenid fam-ily Petalomonidae; this conclusion was based on theappearance of a rigid cell containing strip-like surface stri-ations [12]. However, C. aureus was subsequently col-lected from low-oxygen sediments in the Santa BarbaraBasin, CA (USA) and partially studied with light and scan-ning electron microscopy (LM and SEM, respectively)[13,14]. These studies demonstrated that like P. mariager-ensis, C. aureus was covered with the rod-shape epibioticbacteria, rather than pellicle strips per se.The ultrastructure and molecular phylogenetic position ofC. aureus is currently unknown. These data are expected toeukaryote symbioses within the group and beyond. Themain goals of this study were to characterize theultrastructure and molecular phylogenetic position of C.aureus using small subunit (SSU) rDNA sequences andtransmission electron microscopy (TEM) of serially sec-tioned cells. Our results demonstrated that C. aureus is thefirst member of a novel group of anoxic euglenozoans –referred to here as the "Symbiontida" – to be characterizedat both the molecular and ultrastructural levels. A com-panion study centered on the molecular identity anddetailed ultrastructure of the epibiotic bacteria on C.aureus is currently underway.MethodsCollection of organismsCalkinsia aureus was collected using a Soutar box corer orMC-800 multi corer from the sea floor sediment (580 –592 m in depth) of the Santa Barbara Basin, California,USA in September of 2007 and June of 2008. Sedimentcore samples were collected on the R/V Robert GordonSproul. Some sediment samples were immediately fixedfor transmission electron microscopy (TEM) with anequal volume of 4% (v/v) glutaraldehyde in 0.2 Msodium cacodylate buffer (SCB) (pH 7.2) and stored at4°C. The remaining sediment samples were stored in 50ml plastic tubes at 4°C and subsequently processed forlight microscopy, scanning electron microscopy (SEM)and DNA extraction.Light and electron microscopyLight micrographs of over 100 living cells were takenusing a Zeiss Axioplan 2 imaging microscope and a LeicaDC500 digital chilled CCD camera.Cells of C. aureus were prepared for SEM by mixing anequal volume of fixative solution containing 4% (v/v) glu-taraldehyde in 0.2 M SCB (pH 7.2) at room temperature.The fixed cells were mounted on polycarbonate Milliporefilters (13-mm diam., 5-μm pore size) or glass platescoated with poly-L-lysine at room temperature for 1 hr.The cells were rinsed with 0.1 M SCB and fixed in 1%osmium tetroxide for 30 min. The osmium-fixed cellswere then rinsed with 0.1 M SCB and dehydrated with agraded ethanol series from 30% to absolute ethanolbefore being critical point dried with CO2 using a Tousi-mis Critical Point Dryer. The dried cells were then coatedwith gold using a Cressington 208HR High ResolutionSputter Coater, and observed with a Hitachi S-4700 fieldemission scanning electron microscope.Cells of C. aureus prepared for TEM were kept in fixativesolution for two months before being individually iso-lated from the surrounding sediment in the sample. Iso-Page 2 of 22(page number not for citation purposes)help establish robust inferences about the overall diversityof euglenozoans and the ultrastructure of prokaryote-lated cells were rinsed with 0.2 M SCB (pH 7.2) threetimes and then fixed in 1% (w/v) osmium tetroxide in 0.2BMC Microbiology 2009, 9:16 http://www.biomedcentral.com/1471-2180/9/16M SCB (pH 7.2) at room temperature for 1 hr before beingdehydrated through a graded series of ethanol and 100%acetone. The dehydrated cells were then infiltrated withacetone-Epon 812 resin mixtures and 100% resin. Indi-vidual cells were flat embedded and serial sectioned in dif-ferent orientations (i.e. transverse and longitudinal).Ultra-thin serial sections were collected on copper, Form-var-coated slot grids and stained with 2% (w/v) uranylacetate and lead citrate [15] before being observed using aHitachi H7600 electron microscope. A total of 899 micro-graphs from 12 different cells were observed.Two different media were used in an attempt to culture C.aureus: 5% of TYGM-9 (ATCC medium 1171) and 5% ofmodified PYNFH medium (ATCC medium 1134), dilutedin anoxic and axenic seawater at 4°C. However, the cellsdid not grow in either medium.DNA extraction, PCR amplification, alignment and phylogenetic analysisTwenty individual cells of C. aureus that corresponded tothe original species description (distinctive color, size,shape and motility [12]) were isolated from the sedimentand washed twice in sterilized seawater. Because of thehighly distinctive morphology of C. aureus and the pre-cautions taken, the possibility of contamination is exceed-ingly low. Genomic DNA was extracted from the cellsusing MasterPure Complete DNA and RNA purificationKit (Epicentre, WI, USA). The polymerase chain reaction(PCR) was performed using a total volume of 25 μl andthe PuRe Taq Ready-To-Go PCR beads kit (GE Healthcare,Buckinghamshire, UK). Nearly the entire SSU rRNA genewas amplified from genomic DNA using eukaryotic uni-versal primers (PF1: 5'-GCGCTACCTGGTTGATCCT-GCCAGT-3' and R4: 5'-GATCCTTCTGCAGGTTCACCTAC-3'). The PCR protocol had an initial denaturation stage at95°C for 2 min; 35 cycles involving 94°C for 45 s (dena-turation), 55°C for 45 s (annealing), and 72°C for 1.5min (extension); and final extension at 72°C for 5 min.The amplified DNA fragments were purified from agarosegels using UltraClean 15 DNA Purification Kit (MO Bio,CA, USA), and then cloned into the TOPO TA Cloning Kit(Invitrogen, CA, USA). The C. aureus sequence was depos-ited in DDBJ/EMBL/GenBank under the accessionnumber EU753419.The SSU rRNA sequence of C. aureus was visually alignedwith taxa representing all of the major groups of eukaryo-tes, forming (i) a 38-taxon alignment with ambiguouslyaligned regions excluded (988 unambiguously alignedpositions). In order to more comprehensively evaluate thephylogenetic position of C. aureus within the Euglenozoa,we analyzed three additional datasets: (ii) a 35-taxonaligned positions); (iii) a 29-taxon alignment of eugleno-zoan sequences including three fast-evolving euglenidsequences – namely Astasia torta (AF403152), Menoidiumbibacillatum (AF247598) and Ploeotia costata (AF525486)– and excluding the short environmental sequences (734unambiguously aligned positions); and (iv) a 25-taxonalignment of euglenozoan sequences excluding both theshort environmental sequences and the fastest-evolvingeuglenid sequences (1025 unambiguously aligned posi-tions). The highly divergent sequences from phagotrophiceuglenids produced a large number of ambiguouslyaligned regions in the 35-taxon and 29-taxon alignments;accordingly, these regions were excluded from our analy-ses.PhyML [16] was used to analyze all four datasets (oneheuristic search per dataset) with maximum-likelihood(ML) using a general-time reversible (GTR) model of basesubstitutions [17] that incorporated invariable sites and adiscrete gamma distribution (eight categories) (GTR + I +G model). The GTR model was selected using the programMrAIC 1.4.3 with PhyML http://www.abc.se/~nylander/mraic/mraic.html. Model parameters were estimated fromeach of the original datasets. ML bootstrap analysis wasconducted with the same settings described above (100pseudoreplicates; one heuristic search per pseudorepli-cate).The four alignments were also analyzed with Bayesianmethods using the MrBayes program [18]. The programwas set to operate with a gamma distribution and fourMonte-Carlo-Markov chains (MCMC) starting from a ran-dom tree. A total of 2,000,000 generations were calculatedwith trees sampled every 50 generations and with a priorburn-in of 100,000 generations (2000 sampled trees werediscarded; burn-in was checked manually). A majorityrule consensus tree was constructed from 38,000 post-burn-in trees. Posterior probabilities correspond to thefrequency at which a given node was found in the post-burn-in trees. Independent Bayesian runs on each align-ment yielded the same results.ArchivingA digital archive of this paper is available from PubMedCentral and print copies are available from libraries in thefollowing five museums: Natural History Museum Library(Cromwell Road, London, SW7 5BD, UK), AmericanMuseum of Natural History (Department of Library Serv-ices, Central Park West at 79th St., New York, NY, 10024,USA), Muséum national d'Histoire naturelle (Directiondes bibliothèques et de la documentation, 38 rue Geof-froy Saint-Hilaire, 75005 Paris, France), Russian Academyof Sciences (Library for Natural Sciences of the RAS Zna-Page 3 of 22(page number not for citation purposes)alignment of euglenozoan sequences and ten relativelyshort environmental sequences (760 unambiguouslymenka str., 11, Moscow, Russia) and Academia SinicaBMC Microbiology 2009, 9:16 http://www.biomedcentral.com/1471-2180/9/16(Life Science Library, 128 Sec. 2 Academia Rd, Nankang,Taipei 115, Taiwan R.O.C.).ResultsGeneral MorphologyCalkinsia aureus ranged from 41.7–71.2 μm long (averagelength = 56.7 μm, n = 32) and from 14.5–23.3 μm wide(average width = 18.3 μm, n = 32). The oval-shaped cellswere distinctively orange in color, dorsoventrally com-pressed, and possessed a tapered tail that was about 10μm long (Figure 1). Two heterodynamic flagella wereinserted within a subapical depression at the anterior endof the cell. The longer anterior flagellum was about twicethe length of the cell and was held straight forward duringgliding. The shorter posterior flagellum was half thelength of the cell and was usually positioned within a ven-tral groove. Colorless rod-shaped epibiotic bacteria wereoriented along the longitudinal axis of the cell (Figures1B–D, 2). The posterior half of the cell usually containedan accumulation of spherical food bodies, some of whichcontained diatom frustules (Figures 1A–F, 3A–B). Cystformation and sexual reproduction were not observed.Asexual reproduction was achieved by cell division alongthe longitudinal axis of the cell. Following the replicationof the flagellar apparatus, a cleavage furrow formed at theanterior end of the cell and advanced toward the posteriorend of the cell (Figure 1E).Cell Surface and Extracellular MatrixThe longitudinally arranged, epibiotic bacteria consistedof only one rod-shaped morphotype (3–5 μm long and0.350 μm wide) that collectively formed a dense coat overthe entire surface of the host cell (Figures 2, 3A, 3C). Atleast 128 epibiotic bacteria were observed in transversesections through one cell of C. aureus (Figure 3C). The cellsurface beneath the epibiotic bacteria consisted of sevenmain layers (from outside to inside): (1) a glycocalyx, (2)a highly organized extracellular matrix, (3) the host cellmembrane, (4) an array of parallel microtubules, (5) adouble-layered lamella, (6) superficially arranged mito-chondrion-derived organelles and (7) cisternae of endo-plasmic reticulum (ER) (Figures 3, 4, 5). The extracellularmatrix surrounded the entire cell except for the inside lin-ing of the vestibulum, which leads to the flagellar pocketand feeding pockets (Figures 2C, 3D–E). The portion ofthe extracellular matrix positioned just inside the openingof the vestibulum lacked epibiotic bacteria and consistedof fine hair-like structures, or somatonemes (Figure 3E).The extracellular matrix beneath the epibiotic bacteria wascoated with a thin glycocalyx (Figures 4B–D, 5). The extra-cellular matrix itself was bright orange, approximately 100nm thick and perforated with hollow tubes that joined theplasma membrane of the host with the glycocalyx beneathAn array of evenly spaced microtubules was positionedimmediately beneath the plasma membrane of the host(Figures 4A, 4C–D, 5). These microtubules were derivedfrom the dorsal lamina (DL) of the flagellar apparatus (seedescription below). Each microtubule was connected toneighboring microtubules with electron dense "arms"(Figure 4D). A double-layered lamella was positionedbetween the layer of microtubules and a deeper layer ofmitochondrion-derived organelles (Figures 4A–B, 4D).The mitochondrion-derived organelles were discoidal inshape, were bounded by two membranes and lackedmitochondrial cristae or inclusions such as kinetoplasts(Figures 4A–B, 4E). Moreover, we did not observe any evi-dence of euglenid-like pellicle features, such as the pres-ence of S-shape proteinaceous strips or discontinuities inthe layer of microtubules.Nucleus, Vestibulum and Associated PocketsAn anterior nucleus was positioned near the ventral sideof the cell and contained a prominent nucleolus and con-densed chromosomes (Figures 3A, 3C–D). The vestibu-lum was positioned directly above the nucleus as thisspace passed from the ventral, subapical opening towardthe dorsal side of the cell (Figure 3C). The vestibulumthen extended posteriorly along the dorsal side of the celland branched into three distinct pockets: (1) a novel"extrusomal pocket", (2) a flagellar pocket and (3) a feed-ing pocket (Figures 3A, 3C; described in more detailbelow). A battery of longitudinally arranged extrusomeswas connected to the base of the extrusomal pocket andwas nested within a notch on the dorsal side of the ventralnucleus (Figures 1B, 3A, 3C). Each extrusome was about160 nm in diam. (Figure 3G). The battery of extrusomeswas indistinguishable from the feeding rods of euglenidswhen viewed with the light microscope, and discharged asa single unit through the anterior opening (Figures 1B,1H). The flagellar pocket was located on the dorsal side ofthe cell and contained two flagella that inserted at the bot-tom of the pocket (Figures 6, 7; described in more detailbelow). The feeding pocket was located to the right of theflagellar pocket and extended horizontally before taperingposteriorly toward the ventral side of the cell (Figures 8, 9;described in more detail below).Flagella, Transition zones and Basal BodiesBoth flagella contained a paraxonemal rod adjacent to theaxoneme, and flagellar hairs were not observed on eitherflagellum (Figure 6A). The paraxonemal rod in the dorsalflagellum (DF) had a whorled morphology in transversesection, and the paraxonemal rod in the ventral flagellum(VF) was constructed of a three-dimensional lattice of par-allel fibers (Figures 6B, 6K). The entire length of theaxoneme had the standard 9+2 architecture of microtu-Page 4 of 22(page number not for citation purposes)the epibiotic bacteria (Figures 1G, 4A–C, 5). bules (Figure 6B). The central microtubules within eachaxoneme terminated approximately 1 μm above the flag-BMC Microbiology 2009, 9:16 http://www.biomedcentral.com/1471-2180/9/16Page 5 of 22(page number not for citation purposes)Differential interference contrast images of the living cell of Calkinsia aureusFigu  1Differential interference contrast images of the living cell of Calkinsia aureus. The micrographs show the distinc-tively orange color of the cell, two flagella, epibiotic bacteria and ingested material. A. Anterior flagellum (AF) and the poste-rior flagellum (PF) inserted into an anterior opening (arrow). The ingested material is present in the middle and posterior regions of the cell. B. Surface striations (arrowhead) and a longitudinal rod-like structure (double arrowhead) indicative of a feeding apparatus. C. AF and PF emerging from the anterior opening. The arrowhead shows striation on the surface of the cell. D. Bacteria (arrowheads) that have disassociated with C. aureus. E. A cell undergoing division showing a longitudinal cleavage furrow starts from the anterior end. The ingested material is present in the middle and posterior regions of the cell. F. Clear cytoplasm extruded from posterior of the cell. G. Bright orange extracellular matrix. H. Bundle of extrusomes (double arrow-head) that have been discharged from extrusomal pocket through the anterior opening. (bars = 10 μm, A-C at same scale).BMC Microbiology 2009, 9:16 http://www.biomedcentral.com/1471-2180/9/16ellar insertion (Figures 6A, 6C); this 9 + 0 arrangement isrecognized as the transition zone in accordance with thedefinition proposed by Hibberd [19]. The transition zoneand basal bodies are further described here from the distalend toward the proximal end.The central space within the proximal half of the transi-tion zone contained three distinct elements: faint spokes(denoted as 'a'), an outer concentric ring positioned justinside the microtubular doublets (denoted as 'b'), andelectron dense globules (denoted as 'c') (Figures 6D, 6L).Each faint spoke extended from a microtubular doublettoward the center of the transition zone. The globuleswere positioned at the intersections of each faint spokeand the outer concentric ring (Figures 6D, 6L). In moreproximal points along the transition zone, nine "radialpresent within the central space when observed in bothlongitudinal and transverse section (Figures 6A, 6F–G).The opaque core consisted of six distinct elements: ninespokes extending from each doublet (denoted as 'a'), theouter concentric ring (denoted as 'b'), nine electron denseglobules associated with the outer concentric ring(denoted as 'c'), a central electron dense hub (denoted as'd'), an inner concentric ring (denoted as 'e') and nineradial connectives extending from each doublet to theflagellar membrane (denoted as 'f') (Figures 6F, 6M). Theradial connectives disappeared just above the distalboundary of the basal body (Figures 6A, 6G), and the ele-ments within the central space disappeared just below thedistal boundary of the basal body (Figures 6A, 6H).The dorsal basal body (DB) and ventral basal body (VB)Scanning electron micrographs (SEM) of Calkinsia aureusFigure 2Scanning electron micrographs (SEM) of Calkinsia aureus. A. The ventral side of C. aureus showing the anterior open-ing, a longitudinal groove and epibiotic bacteria. B. The dorsal side of the C. aureus showing the epibiotic bacteria. (A, B bars = 10 μm). C. High magnification SEM of the anterior vestibular opening showing the absence of epibiotic bacteria on the extra-cellular matrix (arrow). (bar = 3 μm).Page 6 of 22(page number not for citation purposes)connectives" extended from each doublet toward the flag-ellar membrane (Figures 6E–F), and an opaque core wasanchored the dorsal flagellum (DF) and ventral flagellum(VF), respectively. Both basal bodies were approximatelyBMC Microbiology 2009, 9:16 http://www.biomedcentral.com/1471-2180/9/16Figure 3 (see legend on next page)Page 7 of 22(page number not for citation purposes)BMC Microbiology 2009, 9:16 http://www.biomedcentral.com/1471-2180/9/161.6 μm long, arranged in parallel to each other, and pos-sessed nine transitional fibers extending from each triplettowards the cell membrane (Figures 6A, 6H–I). Internalcartwheel elements were present within the most proxi-mal ends of both basal bodies (Figures 6J, 7G).Flagellar Root SystemThe flagellar root system is described here from the proxi-mal boundary of the basal bodies toward the distalboundary of the basal bodies as viewed from the anteriorend of the cell (Figure 7). The DB and the VB were joinedwith a connecting fiber and associated with three microtu-bular roots: the dorsal root (DR), the intermediate root(IR) and the ventral root (VR) (Figures 7A–B). The VB, IRand VR were also associated with three fibrous roots: theright fiber (RF), the intermediate fiber (IF) and the leftfiber (LF) (Figure 7B). The DR and IR were associated withtwo thin laminae: the dorsal lamina (DL) and the IR-asso-ciated lamina (IL), respectively (Figures 7A–D, 9B).The DR originated from the dorsal side of the DB and con-sisted of three microtubules near the proximal boundary.Both the DR and the DL extended toward the anterior sideof the cell (Figures 7B–D) and supported the flagellarpocket (Figures 7E–F). The DR occupied the dorsal leftside of the flagellar pocket; the DL occupied the dorsalright side of the flagellar pocket and extended from the VRto the DR at the level of the transition zone (Figures 7E–F). A row of linked microtubules (LMt) originated in closeassociation with the DL (above the VR) and supported theright side of the flagellar pocket (Figures 7F, 7H). The DLand LMt extended from the left side of the flagellar pocketto the right side near the posterior boundary of the vesti-bulum (Figures 8A–E). The LMt supported the inner lin-ing of the vestibulum, turned posteriorly along the curveformed by the ventral opening (Figure 3E) and ultimatelybecame the sheet of microtubules located beneath theplasma membrane of the entire cell (Figures 4A, 4C–D).of four microtubules near the proximal boundary (Figures7B–C, 7G). The left side of the IR was tightly associatedwith the IL and two fibrous roots: the LF and the IF (Figure7B). The LF extended laterally and was about 500 nmlong; the IF extended to the left ventral side of the cell andwas about 1.5 μm long (Figures 7B–C). The IL was associ-ated with the left side of the IR along its entire length, andthe IR and IL became more closely associated as theyextended anteriorly along the left side of the flagellarpocket (Figures 7I–K). The microtubules from the IR even-tually merged with the left side of the LMt-DL and likelycontributed to the sheet of microtubules located beneaththe plasma membrane of the entire cell (Figures 8A–C).The VR originated from the ventral side of the VB and con-sisted of nine microtubules that were closely associatedwith the RF (Figures 7A, 7G). The RF extended toward theright-ventral side of the cell and was about 1 μm long (Fig-ures 7A–C). The microtubules from the VR supported theright side of the flagellar pocket and joined the right sideof the LMt and the DL (Figures 7D–F, 7L). The microtu-bules from the VR ultimately became one of the elementsthat reinforced the feeding apparatus (Figures 8, 9).Feeding ApparatusThe feeding apparatus was positioned on the right side ofthe flagellar pocket and is described here along the poste-rior to anterior axis. This apparatus consisted of four mainelements or spaces: a feeding pocket, a VR embeddedwithin six electron-dense fibers, a compact "oblique stri-ated fiber" (OSF) and a "congregated globule structure"(CGS) (Figures 8, 9C). The OSF was approximately 1.5μm long, 800 nm wide and 500 nm high and was posi-tioned between the feeding apparatus and the right side ofthe flagellar pocket (Figures 8A, J). The CGS attached tothe anterior side of the OSF (Figures 8B–E, 8J). Six rows ofelectron dense fibers, each containing a microtubule fromthe VR, passed over the top of the OSF-CGS complex (Fig-ures 8C–F, 8J–K). The VR and the six associated fibersTransmission electron micrographs (TEM) showing the general morphology of Calkinsia aureusFigure 3 (see previous page)Transmission electron micrographs (TEM) showing the general morphology of Calkinsia aureus. A. Sagittal TEM showing the nucleus (N) with condensed chromatin and a conspicuous nucleolus (Nu), a battery of extrusomes (E), the vesti-bulum (V) located on the dorsal side of the cell, ingested material and epibiotic bacteria on the extracellular matrix. The extru-somal pocket (EP) branched from the vestibulum (V) (bar = 4 μm). B. Ingested material containing diatom frustules (arrow). (bar = 2 μm). C. Cross section of the cell through the nucleus (N), the battery of extrusomes (E), the flagellar pocket (FLP) and the feeding pocket (FdP). (bar = 2 μm). D. High magnification view through the vestibulum (V) that is opened on the ventral side of the cell. E. High magnification view through the anterior opening showing the termination of the extracellular matrix (double arrowhead) and fine somatonemes (S) or hair-like structures on the perforated matrix (arrows) that is not covered with epibiotic bacteria. The arrowhead indicates the supportive microtubular sheet that lines the inside of the cytostome and turns along the cell surface. (D, E, bars = 1 μm). F. Hairs (arrow) on the wall of the vestibulum (V). (bar = 1 μm). G. Cross section showing the battery of tubular extrusomes (E). (bar = 2 μm).The IR was positioned between the two basal bodies, orig- reinforced the anterior-right side of the feeding pocketPage 8 of 22(page number not for citation purposes)inated from the right dorsal side of the VB, and consisted (Figures 8C–E). The left side of the feeding pocket wasBMC Microbiology 2009, 9:16 http://www.biomedcentral.com/1471-2180/9/16Page 9 of 22(page number not for citation purposes)Transmission electron micrographs (TEM) showing the surface ultrastructure of Calkinsia aureusFigure 4Transmission electron micrographs (TEM) showing the surface ultrastructure of Calkinsia aureus. A. Tangential TEM section showing conduit-like perforations (arrowheads) embedded within the extracellular matrix (Ex), an array of micro-tubules, and mitochondrion-derived organelles (MtD). (bar = 1 μm). B. Mitochondrion-derived organelles (MtD) with two membranes (arrow) above the ER. The convoluted appearance of the cell plasma membrane (double arrowhead) and a longitu-dinal view of a microtubule (arrowhead) are also shown. A glycocalyx (GL) covers the surface of the extracellular matrix (Ex). C. Transverse TEM showing the epibiotic bacteria (B), the glycocalyx (GL), a conduit-like perforation (arrow) through the extracellular matrix (Ex) and the underlying sheet of microtubules (B, C, bars = 500 nm). D. High magnification view showing the epibiotic bacteria (B), the glycocalyx (GL), the extracellular matrix (Ex), the cell plasma membrane (double arrowhead), and the double-layered structure (arrowhead; derived from the dorsal lamina) beneath a sheet of inter-connected microtubules (bar = 200 nm). E. Mitochondrion-derived organelles (MtD) (bar = 500 nm). Inset: High magnification TEM showing the two membranes that surround the mitochondrion-derived organelles (width of inset = 400 nm).BMC Microbiology 2009, 9:16 http://www.biomedcentral.com/1471-2180/9/16reinforced by a striated fiber that extended from the leftside of the CGS (Figures 8E–F, 8K, 9C).The feeding pocket was surrounded by an accumulationof small vesicles and branched from the vestibulumtoward the ventral side of the cell before turning towardthe posterior end of the cell (Figures 8A–D, 9C). Serialoblique sections through the feeding pocket did not dem-onstrate distinctive feeding vanes or rods per se; only theVR microtubules within the electron dense fibers wereobserved (Figure 8H). Nonetheless, the vestibular junc-tion (or crest) between the flagellar pocket and the feedingpocket contained a "tomentum" [20] of fine hairs (Figure8I).Molecular Phylogenetic Position as Inferred from SSU rDNAWe determined the nearly complete sequence of the SSUrRNA gene of C. aureus (2034 bp). Maximum likelihood(ML) analyses of (i) a 38-taxon alignment including rep-resentative sequences from the major lineages of eukaryo-tes, robustly grouped the sequence from C. aureus with theEuglenozoa (e.g. Euglena, Diplonema and Trypanosoma)(Figure 10). In order to  more comprehensively evaluatethe phylogenetic position of C. aureus within the Eugleno-zoa, we analyzed three additional datasets: (ii) a 35-taxonalignment (Figure 11), (iii) a 29-taxon alignment (Addi-file 2) (see Methods for details).Tree topologies of these three ML analyses were very sim-ilar (Figure 11, Additional Files 1, 2). Accordingly, theresults from the analyses of the 35-taxon dataset includingseveral short environmental sequences, was an accuraterepresentation of all three analyses (Figure 11). Eugleno-zoan sequences clustered into five major groups with highstatistical support: a kinetoplastid clade, a diplonemidclade, a bacteriovorous euglenid clade (i.e. Notosolenusand Petalomonas), a clade consisting of eukaryovorous andphotosynthetic euglenids, and a novel clade referred tohere as the "Symbiontida". The relationships among theseclades (i.e. the backbone) were not resolved (Figure 11).Additional phylogenetic analyses using alternative out-groups (e.g., heteroloboseans) recovered the same basictree topology shown in Figure 11: (1) Calkinsia aureus is amember of a distinct euglenozoan subclade consisting ofsequences derived from environmental PCR surveys, and(2) this clade is not convincingly affiliated with any oneof the three known euglenozoan subgroups (euglenids,kinetoplastids and diplonemids). Moreover, the sequencefrom C. aureus occupied the deepest position within theSymbiontida, which otherwise consisted of seven envi-ronmental sequences collected from Northern Europeand South America (Figure 11).Diagram of the cell surface of Calkinsia aureusFigure 5Diagram of the cell surface of Calkinsia aureus. The diagram shows epibiotic bacteria (B), the glycocalyx (GL), the perfo-rated extracellular matrix (Ex), the host cell plasma membrane (double arrowhead), the linked microtubules (LMt), the double-layered structure (arrowhead), mitochondrion-derived organelles (MtD) and cisternae of endoplasmic reticulum (ER).ERMtDLMtExBGLPage 10 of 22(page number not for citation purposes)tional file 1), and (iv) a 25-taxon alignment (AddtionalBMC Microbiology 2009, 9:16 http://www.biomedcentral.com/1471-2180/9/16DiscussionSeveral poorly studied flagellates, some with discoidal-shaped mitochondrial cristae, have, at one time oranother, been suspected to be close relatives of eugleno-zoans (e.g. Stephanopogon, Hemimastix, Bordnamonas, Cryp-taulax, Postgaardi and Calkinsia) [21-24]. The bestparaxonemal rods (i.e. a whorled structure in the DF andthree-dimensional lattice of parallel fibers in the VF), and(3) tubular extrusomes [9]. The presence of theseultrastructural features in very diverse lineages of flagel-lates, in combination with molecular phylogenetic data,has established the identity and composition of theTransmission electron micrographs (TEM) showing paraxonemal rods in the flagella, the flagellar transition zone and the basal bodies of Calkinsia aureusFigure 6Transmission electron micrographs (TEM) showing paraxonemal rods in the flagella, the flagellar transition zone and the basal bodies of Calkinsia aureus. A. Longitudinal section of the dorsal flagellum (DF) showing the flagellar transition zone and the dorsal basal body (DB) (bar = 500 nm). B-J. Non-consecutive serial sections through the DF (B), the flagellar transition zone (C-G), and the DB (H-J) as viewed from anterior end (images at same scale, bar = 200 nm). B. Section showing the 9+2 configuration of axonemal microtubules and the tubular paraxonemal rod (arrow) in the DF. C. Section showing termination of central microtubules and the 9+0 configuration of axonemal microtubules. D. Section showing the transition zone through an outer concentric ring associated with nine electron dense globules inside of each doublet and faint spokes that extend inward from the each globule (see L for a diagram of this micrograph). E. Section through the nine radial connectives (arrowhead) that extend outward from each doublet to the flagellar membrane. F. Section showing the radial con-nectives that extend outward toward the flagellar membrane, the spokes that extend inward from the microtubular doublets, the central electron dense hub, and inner concentric rings (see M for the diagram of this micrograph). G. Section showing the electron dense hub and inner and outer concentric rings, and the absence of radial connectives. H. A section at the level of the insertion of the DF. The transitional fibers (double arrowheads) extending from the microtubular triplets of the DB are shown. I. Section through the area just below the distal boundary of the DB. The transitional fibers (double arrowheads) connect to each microtubular triplet. J. Section through the proximal region of the DB showing the cartwheel structure. K. View through the paraxonemal rod of the ventral flagellum (VF) (bar = 500 nm). L. Diagram of the level of D showing faint spokes (a) that extend inward from each globule, an outer concentric ring (b) and nine electron dense globules (c). M. Diagram of the level of F showing spokes (a), an outer concentric ring (b), nine electron dense globules (c), an electron dense hub (d), an inner con-centric ring (e) and radial connectives (f).Page 11 of 22(page number not for citation purposes)synapomophies for the Euglenozoa are (1) a tripartiteflagellar root system (DR, IR and VR), (2) heteromorphicEuglenozoa [7,9].BMC Microbiology 2009, 9:16 http://www.biomedcentral.com/1471-2180/9/16Figure 7 (see legend on next page)Page 12 of 22(page number not for citation purposes)BMC Microbiology 2009, 9:16 http://www.biomedcentral.com/1471-2180/9/16Calkinsia aureus was originally described as a member ofthe Euglenida with light microscopical information [12],and we demonstrate here that these flagellates possess allthree ultrastructural synapomorphies for the Euglenozoa.Moreover, the permanently condensed chromatin, longflagellar transition zone, longitudinal cell division andlong basal bodies are also features found in many othereuglenozoans [25]. These morphological data were con-cordant with our comparative analyses of SSU rDNAshowing that C. aureus is robustly embedded within theEuglenozoa clade (Figures 10, 11). However, C. aureuslacked traits that are specific to any of the three previouslyrecognized euglenozoan subgroups (e.g., kinetoplasts,pellicle strips, or absence of paraxonemal rods). Thefaintly striated pellicle originally attributed to C. aureususing light microscopy is, in actuality, the longitudinallyarranged rod-shaped epibiotic bacteria [13,14]. The sheetof microtubules beneath the plasma membrane in C.aureus was continuous over the entire cell, like in kineto-plastids and diplonemids, rather than interrupted by peri-odic discontinuities like in euglenids [26-28] (Figure 3C).There was also no clear evidence of a euglenid-like feedingapparatus consisting of rods and vanes [20,26,29].Accordingly, our study indicates that with present knowl-edge, C. aureus should not be considered a member of theEuglenida or more specifically, a member of the Petalo-monadidae as originally classified [12].Absence of Mitochondria with CristaeAerobic kinetoplastids and euglenids possess well-devel-oped discoid-shaped cristae within their mitochondria[26], and diplonemids and Hemistasia possess a few flat-shaped cristae within each mitochondrion [30-32]. Bycontrast, both C. aureus and P. mariagerensis lack recogniz-in morphology to the well-studied hydrogenosomesdescribed in other anoxic flagellates (e.g. Trichomonas)[33]. Hydrogenosomes are the descendents of mitochon-dria and function to produce molecular hydrogen, acetate,CO2 and ATP in anoxic environments [34,35]. A moreconfident functional characterization of the mitochon-drion-derived organelles in C. aureus or Postgaardi willrequire biochemical and molecular biological assays.A Novel Extracellular MatrixThe plasma membrane of C. aureus was reinforced with acontinuous sheet of microtubules and a double-layeredlamella, which was in turn subtended by a dense array ofmitochondrion-derived organelles (Figures 4, 5). Thisoverall organization, where mitochondrion-derivedorganelles are located immediately beneath a sheet of sur-face microtubules, has also been observed in Postgaardi.However, a uniform and perforated extracellular matrixenveloped the cell surface of C. aureus, and so far as weknow, the organization of this cell covering is novel notonly among euglenozoans, but also among eukaryotes(Figures 4, 5). Because both the epibiotic bacteria and thehost cell cytoplasm were colorless (Figures 1D, 1F–G), thedistinctively orange color of C. aureus is clearly attributa-ble to the chemical composition of the extracellularmatrix (Figure 1G). Moreover, the even distribution oftiny tubes within the matrix provide conduits between thehost plasma membrane and the epibiotic bacteria andpresumably facilitate metabolic exchanges necessary forsurvival in low-oxygen environments. This interpretationis consistent with knowledge of anoxic ciliates, which alsomaintain an intimate physical relationship between mito-chondrion-derived organelles (immediately beneath thehost plasma membrane) and epibiotic bacteria (immedi-ately above the host plasma membrane) [36,37].Transmission electron micrographs (TEM) showing the organization of microtubular roots that originate from the dorsal and ventral basal bodies (DB and VB, respectively)Figure 7 (see previous page)Transmission electron micrographs (TEM) showing the organization of microtubular roots that originate from the dorsal and ventral basal bodies (DB and VB, respectively). Those are viewed from the anterior end (A-F at same scale, bar = 500 nm). A. The proximal region of the basal bodies close to the cartwheel structure. The dorsal root (DR) origi-nates from the DB; the intermediate root (IR) and the ventral root (VR) extend from the VB. A dorsal lamina (DL) attaches to the dorsal side of the DR; the right fiber (RF) is close to the ventral side of the VR. B. Section showing the right fiber (RF), the IR-associated lamina (IL), a left fiber (LF) and an intermediate fiber (IF) associated with the VB. The arrow points to the connec-tive fiber between the DB and the VB. Dense fibrils (double arrowhead) extend to the right side of each microtubule of the intermediate root (IR). C. Section through the middle part of the DB and the VB. D. Section through the insertion of the flag-ella. E. Section through the flagellar transition zone showing the extension of the DL and disorganization of the VF. F. Section showing the linked microtubules (LMt) associated with the dorsal lamina (DL) and the ventral root (VR). G. High magnification view of proximal area of the two basal bodies, the DB and the VB, of A showing the cartwheel structure and the dorsal lamina (DL) on the dorsal side of the dorsal root (DR). The double arrowhead indicates the fibril from each microtubule of the IR. H. High magnification view of right wall of the pocket of F showing the LMt and the DL. I. High magnification view of the IR of D showing the relationship among the IR, IL and IF. J. High magnification view of the IR at the level of E showing the IR and IL. K. High magnification view of the IR and IL. L. High magnification view of the VR in E. (G-L, bars = 200 nm).able mitochondria with cristae, and instead, contain dou-Page 13 of 22(page number not for citation purposes)ble-membrane bound organelles that are nearly identicalBMC Microbiology 2009, 9:16 http://www.biomedcentral.com/1471-2180/9/16Figure 8 (see legend on next page)Page 14 of 22(page number not for citation purposes)BMC Microbiology 2009, 9:16 http://www.biomedcentral.com/1471-2180/9/16Flagellar ApparatusThe flagella of most euglenids and kinetoplastids havenon-tubular mastigonemes (or flagellar hairs) that,among other functions, facilitate gliding motility [38];however, these structures are absent in C. aureus, P. mar-iagerensis and diplonemids. Instead, a tomentum of finehairs are present at the crest of the feeding pocket in C.aureus that are similar to those described in the pho-totrophic euglenid Colacium [39], the phagotrophic eugle-nid Peranema [40], and the kinetoplastid Cryptobia[41,42]. Although hairs associated with the feeding pockethave not been routinely observed in other members of theEuglenozoa, the presence of these hairs in distantlyrelated lineages indicate that this feature might representan ancestral state for the Euglenozoa as a whole.Transmission electron micrographs (TEM) of Calkinsia aureus showing the feeding apparatusFigure 8 (see previous page)Transmission electron micrographs (TEM) of Calkinsia aureus showing the feeding apparatus. The ventral flagel-lum was disorganized in all sections (A-D at same scale, bar = 1 μm; E-G at same scale, bar = 1 μm). A. Section showing the oblique striated fibrous structure (OSF) and the VR along the wall of the flagellar pocket (FLP). Arrow points out the LMt and the DL. B. Section through the congregated globular structure (CGS), the OSF and the feeding pocket (FdP). The VR extends to the right. The arrow points out the LMt and the DL, which extend from the VR to the IR and support the dorsal half of the FLP. C. Section showing the VR over the CGS. Arrows show the LMt and DL. D. The VR crosses over the CGS and extends to right side of the FdP. Most of the wall of the FLP is supported by the LMt and DL (arrows). E. A striated fiber (double arrowhead) supports the left side of the FdP and extends from the left side of the CGS. Arrows indicate the extension of the LMt and DL. F. Section through the beginning of the vestibulum (V) and the striated fiber (double arrowhead). G. The V is enlarged and the CGS remains at both sides of the FdP. H. High magnification of FdP. I. Tangential TEM section showing the VR with an electron dense fiber along the feeding pocket and a tomentum (T) of fine hairs. J. Longitudinal section through the CGS and the OSF. Six ventral root microtubules embedded within the electron dense fibers (arrowheads). K. High magnification view of the VR supporting the FdP shown in F. Double arrowhead indicates the striated fiber and the six arrowheads indicate the electron dense fibers of the VR. (H-K, bars = 500 nm).Diagram of the cell (A), the flagellar apparatus (B) and the feeding apparatus (C) of Calkinsia aureus based on serial TEM sec-tionsFigure 9Diagram of the cell (A), the flagellar apparatus (B) and the feeding apparatus (C) of Calkinsia aureus based on serial TEM sections. A. Illustration of the cell viewed from the left side; arrow marks the extrusomal pocket. Boxes B and C indicate the plane of view shown in Figures B and C, respectively. B. Illustration of the flagellar apparatus as viewed from left side. C. Illustration of the feeding apparatus as viewed from anterior-ventral side. The double arrowhead marks the striated fiber along the feeding pocket (FdP). Note DL, IF, IL, LF, LMt, and RF are not shown on this diagram for clarity.Page 15 of 22(page number not for citation purposes)BMC Microbiology 2009, 9:16 http://www.biomedcentral.com/1471-2180/9/16The structure of the flagellar transition zone is variableamong kinetoplastids and euglenids, particularly inregard to the presence/absence of peripheral elements andtransition zone [30,32,42], while euglenids only possessthe proximal transitional plate. Although the transitionzone of most euglenids is also hollow, the transition zonePhylogenetic position of Calkinsia aureus within eukaryotes as inferred from SSU rRNA gene sequencesFigure 10Phylogenetic position of Calkinsia aureus within eukaryotes as inferred from SSU rRNA gene sequences. Maxi-mum likelihood (ML) analysis of 38 taxa sampled from phylogenetically diverse eukaryotes. This tree is rooted with opisthokont sequences. ML bootstrap values greater than 50% are shown. Thick branches indicate Bayesian posterior probabil-ities over 0.95. GenBank accession numbers of the sequences analyzed are shown in parentheses.MalawimonasRhodophytaJakobidsAlveolataJakobidsStramenopileHaptophytaGlaucophytaCryptophytaViridiplantaeOpisthokontsCercozoaApusozoaParabasalidsAmoebozoaEuglenozoaOxymonadsTrimastixFornicataHeterolobosea0.1Saccharomyces cerevisiae (V01335) Diaphanoeca grandis (DQ059033) Euglypha rotunda (X77692)Cercomonas sp. (U42449)Apusomonas proboscidea (L37037)Cyanophora paradoxa (X68483)Guillardia theta (X57162)Chilomonas paramecium (L28811)Emiliania huxleyi (AF184167)Pavlova salina (L34669) Chlorella vulgaris (X13688)Zea mays (AF168884)Labyrinthuloides minuta (L27634)Thalassionema nitzschioides (X77702)Porphyra umbilicalis (AB013179) Porphyra miniata (L26200) Paramecium tetraurelia (X03772)Toxoplasma gondii (U03070) Prorocentrum micans (AJ415519) Trimastix pyriformis (AF244903)Streblomastix strix (AY188885)Malawimonas jakobiformis (AY117420)Andalucia godoyi (AY965865)Andalucia incarcerata (AY117419)Diplonema ambulator (AF380996)Euglena gracilis (AF283308)Trypanosoma sp. (EF375883)Calkinsia aureus (EU753419)Jakoba libera (AY117418) Reclinomonas americana (AY117417) Dictyostelium discoideum (K02641)Entamoeba histolytica (X56991)Dysnectes brevis (AB263123)Retortamonas sp. (AF439347)Naegleria gruberi (M18732)Tetramitus thorntoni (X93085)Trichomonas vaginalis (U17510) Tritrichomonas foetus (AY055799) 10094100949694100841009810060100100100100Page 16 of 22(page number not for citation purposes)transitional plates. Kinetoplastids and diplonemids pos-sess distal and proximal transitional plates and a hollowin some euglenids, such as Entosiphon applanatum andNotosolenus (Petalomonas)mediocanellata, has been shownBMC Microbiology 2009, 9:16 http://www.biomedcentral.com/1471-2180/9/16to be electron dense. However, the detailed structure ofthese transition zones still remains to be characterized indetail [29,43]. The central area of the transition zone in C.aureus is also electron dense and contains a complex sys-tem of elements that have never been observed in anyother Euglenozoan so far (Figure 6). Characterization ofthe flagellar transition zone in Postgaardi might demon-strate several homologous elements that would help tofurther establish a close relationship between this lineageand C. aureus.Nonetheless, Diplonema ambulator, Rhynchopus euleeides,R. coscinodiscivorus and C. aureus all have fibers that extendfrom each microtubular doublet to the flagellar mem-brane; these fibers have been called "transitional fibers"[30,32,44]. "Transitional fibers" has also been used todescribe fibers that extended from each microtubular tri-plet of a basal body to the flagellar membrane, which ispotentially confusing [45-47]. Nonetheless, the "radialconnectives" extending from the doublets in the transi-tion zone of C. aureus are nearly identical, and likelyPhylogenetic position of Calkinsia aureus within euglenozoans as inferred from SSU rRNA gene sequencesFigure 11Phylogenetic position of Calkinsia aureus within euglenozoans as inferred from SSU rRNA gene sequences. Maximum likelihood (ML) analysis of 35 taxa focusing on the position of Calkinsia aureus within the Euglenozoa clade. Two jako-bids, Andalucia incarcerata and A. godoyi, are used as outgroups in this analysis. ML bootstrap values greater than 50% are shown. Thick branches indicate Bayesian posterior probabilities over 0.95. Ba, bacteriotroph; Eu, eukaryotroph; Ph, phototroph. Gen-Bank accession numbers of the sequences analyzed are shown in parentheses.EuglenozoaEuglenidaKinetoplastidaDiplonemidaSymbiontidaBaEuPhJakobids (outgroup)0.1Andalucia incarcerata (AY117419)Andalucia godoyi (AY965865)Notosolenus ostium (AF403159)Petalomonas cantuscygni (AF386635)LC23 5EP 5 (DQ504350) [Lost City, Mid-Atlantic]Rhynchopus sp. (AF380997)Diplonema ambulator (AF380996)Diplonema papillatum (AF119811)LC22 5EP 17 (DQ504321) [Lost City,Mid-Atlantic]LC22 5EP 32 (DQ504349) [Lost City,Mid-Atlantic]Peranema trichophorum (AF386636)Eutreptiella gymnastica (AF081590)Euglena stellata (AF081590)Euglena gracilis (AF283308)Discoplastis spathirhyncha (AJ532454)Colacium sp. (DQ140154)Phacus aenigmaticus (AF283313)Monomorphina sp. (DQ140130)Dinema sulcatum (AY061998)Anisonema acinus (AF403160)"Peranema sp." (AY048919)Calkinsia aureus (EU753419) [Santa Barbara Basin, California]CAR_E220 (AY256285) [Cariaco Basin, Venezuela]T53F7 (AY882489)  [Cariaco Basin, Venezuela] CAR_H25  (AY256209) [Cariaco Basin, Venezuela]M4 18D10 (DQ103806) [Mariager Fjord, Denmark]M4 18E09 (DQ103807) [Mariager Fjord, Denmark]FV36 2E04 (DQ310359)  [Framvaren Fjord Norway]FV23 2D3C4 (DQ310255) [Framvaren Fjord, Norway]Trypanosoma sp. (EF375883)Trypanosoma evansi (AY904050)Neobodo designis (AF209856)Bodo saltans (AY998648)Rhynchomonas nasuta (AY998642)Dimastigella mimosa (DQ207576)100100100100100951005069708653100996594 691001009575541007758Page 17 of 22(page number not for citation purposes)homologous, to the 'transitional fibers' extending fromthe doublets in diplonemids, such as D. ambulator.BMC Microbiology 2009, 9:16 http://www.biomedcentral.com/1471-2180/9/16Feeding ApparatusEach of the euglenozoan subgroups contains memberswith an elaborate feeding apparatus [20,26,29,39]. Mostphagotrophic euglenids, for instance, have a distinctivefeeding apparatus consisting of 4–5 central vanes and 2–3 supporting rods [28,48,49]. Some bacteriovorous eugle-nids (e.g. Petalomonas), however, possess a much simplerfeeding apparatus that is very similar to the MTR feedingpockets found in many kinetoplastids (e.g. Bodo) [26].The microtubules that support the rods in phagotrophiceuglenids and the MTR pockets in bacteriovorouseuglenozoans originate from the ventral root of the ven-tral basal body. Similarly, the feeding pocket in C. aureuswas also supported by microtubules that originated fromthe ventral root and is almost certainly homologous to theMTR pockets or rods found in other euglenozoans,including Postgaardi [33]. Moreover, the compact"oblique striated fiber" (OSF) that reinforces the feedingpocket in C. aureus was similar to the amorphous matrixfound in some euglenid feeding rods and might representa vestige of a more elaborate ancestral state. However, thisinference will require improved understanding of themorphological diversity and phylogeny of other eugleno-zoans that are more closely related to C. aureus.A Novel Extrusomal PocketAlthough tubular extrusomes are not widespread withinthe Euglenozoa, several members from each main sub-group possess them, such as the euglenid Entosiphon[50,51]; the kinetoplastids Rhynchobodo [52], Hemistasia[31], and Phylomitus [53]; the diplonemid Diplonema nig-ricans [54]; and Postgaardi mariagerensis [33]. Calkinsiaaureus not only had tubular extrusomes like the lineageslisted above, but they were clustered together much likethe single battery of tubular extrusomes found in Hemista-sia [31]. By contrast, Postgaardi and Rhynchobodo possessseveral smaller batteries of tubular extrusomes that aredispersed throughout the cytoplasm [33,52].The battery of tubular extrusomes in C. aureus wasanchored to a novel extrusomal pocket that branched offof the vestibulum separately from the feeding apparatusand the flagellar apparatus (Figures 3A, 3C, 9). This bat-tery of extrusomes was often discharged as a single unitfrom the extrusomal pocket and through the anterioropening (Figure 1H). The functional significance of thisprocess is unclear.The phagotrophic euglenid Dinema sulcatum also containsa flagellar pocket and reportedly has two additional pock-ets: (1) a "normal" feeding apparatus consisting of sup-portive rods and vanes and (2) an "extra" pocketconsisting of MTR-like microtubules [43]. One previously"extra" pocket is a remnant of the MTR feeding pocketpresent in the ancestral euglenozoan and the rod-and-vane based feeding apparatus represents a duplicated, andgreatly embellished, MTR pocket that arose within aderived lineage of phagotrophic euglenids [7,27,55]. Thishypothesis is consistent with comparative morphologicaldata that indicates other euglenid cytoskeletal compo-nents also evolved by duplication, such as the totalnumber of pellicle strips around the cell periphery[7,28,56,57]. Nonetheless, the extrusomal pocket in C.aureus was supported by the LMt (connected to the dorsalroot) rather than microtubules from the ventral root,which support both MTR pockets and rod-and-vane basedfeeding apparatuses in euglenozoans. Therefore, the extru-somal pocket in C. aureus appears to be novel and doesnot seem to be homologous to any type of feeding appa-ratus reported so far (e.g. a rod-and-vane based appara-tuses or a remnant or duplicated MTR pocket).Euglenozoans with Epibiotic BacteriaPostgaardi mariagerensis [33,58], Euglena helicoideus [59],Dylakosoma pelophilum [60], C. aureus [13] and five uni-dentified euglenozoans from low oxygen environments[13,14,61] have been reported to possess epibiotic bacte-ria on the cell surface. The epibiotic bacteria on D. pelo-philum are spherical, and those on the other taxa are rod-shaped and densely packed on the cell surface. Only oneof the five unidentified euglenozoans, namely "morpho-type C" from Monterey Bay, was studied with both SEMand TEM [61]. The rod-shape epibiotic bacteria on thesecells were not associated with a superficial distribution ofmitochondrion-derived organelles (e.g., hydrogeno-somes) beneath the host plasma membrane. Nonetheless,morphotype C was clearly a euglenid, because the flagellacontained paraxonemal rods, the feeding apparatus con-sisted of rods and vanes, and thin proteinaceous stripssupported the cell surface.By contrast, the combination of ultrastructural features inC. aureus and P. mariagerensis make these lineages difficultto place within the Euglenozoa. Both lineages lack evi-dence of pellicle strips or kinetoplasts and possess parax-onemal rods, tubular extrusomes, mitochondrion-derivedorganelles beneath the plasma membrane, and con-densed chromatin. Detailed comparisons of the feedingapparatus in C. aureus, P. mariagerensis, and other anoxiceuglenozoans should help better establish their phyloge-netic relationships with each other; however, except for C.aureus, this information is currently lacking for nearly allof these lineages, including P. mariagerensis.Molecular Phylogenetic Framework for Euglenozoans in Low-Oxygen EnvironmentsPage 18 of 22(page number not for citation purposes)proposed hypothesis for the presence of two feeding pock-ets in D. sulcatum involves the following inferences: theThe morphology of C. aureus (e.g. the flagellar apparatusand tubular extrusomes) was completely concordant withBMC Microbiology 2009, 9:16 http://www.biomedcentral.com/1471-2180/9/16the molecular phylogenetic data in so far as strongly plac-ing C. aureus within the Euglenozoa, but not with any ofthe three previously recognized subclades. Figure 11shows the phylogenetic position of C. aureus within theEuglenozoa, which consisted of five main clades.Although Petalomonas and Notosolenus branched togetheras a separate clade, morphological evidence strongly sup-ports their inclusion within the Euglenida. Therefore, themolecular phylogenetic data coupled with the morpho-logical data allows us to recognize four clades of eugleno-zoans: the Euglenida, the Kinetoplastida, theDiplonemida and a novel clade of anoxic euglenozoans,hereby named the Symbiontida.The Symbiontida includes several environmentalsequences that were originally designated either asdiplonemid sequences (e.g. T53F7) [62], as unculturedeuglenozoan sequences (e.g. M4 18E09, M4 18D10, FV232D3C4 and FV36 2E04) [63,64] or as "possible earlybranching eukaryotes" (CAR_H25 and CAR_E220) [65].Some of the environmental sequences within the Symbi-ontida were already suspected to represent either a novelsister clade to the Euglenozoa or novel subclade ofeuglenozoans [64]. Nonetheless, we have demonstratedthat the Symbiontida contains several more environmen-tal sequences collected from different low-oxygen envi-ronments and also C. aureus, which provides anorganismal anchor (i.e. the cellular identity) for this clade.We should also note that some environmental sequencesfrom mid-Atlantic hydrothermal vent environments inthe "Lost City", namely LC23 5EP 5, LC22 5EP 17, andLC22 5EP 32, grouped strongly with the diplonemid cladeand not with the Symbiotida [66]. Moreover, the lack ofphylogenetic signal and perhaps also long-branch-attrac-tion were the likely reasons for why the relatively fast-evolving sequences from Notosolenus and Petalomonas didnot cluster strongly with the euglenid clade in our analy-ses of the dataset containing the shortest sequences (Fig-ure 11). Our analysis of the dataset including only thelongest sequences, by contrast, clustered Notosolemus andPetalomonas with all other euglenids, albeit without strongstatistical support (Additional File 2) [67,68].The Symbiontida: A Novel Subclade of the EuglenozoaBefore C. aureus had been studied at the ultrastructuraland molecular phylogenetic levels, one author classifiedthis lineage with P. mariagerensis within the taxon "Post-gaardea" on the basis of microaerophily [10,11].Although our characterization of C. aureus has demon-strated epibiotic bacteria and mitochondrion-derivedorganelles like those described in P. mariagerensis, thepresence of these characters in both lineages does not nec-drion-derived organelles have been found in many differ-ent lineages of anoxic microeukaryotes, such as ciliates,oxymonads, parabasalids, heteroloboseans and eugleno-zoans [36,69]. Moreover, the presence of tubular extru-somes in both C. aureus and P. mariagerensis could be asymplesiomorphic state inherited from a very distanteuglenozoan ancestor.Nonetheless, our phylogenetic analyses demonstrate thatC. aureus is a member of a newly recognized clade ofanoxic euglenozoans consisting mainly of environmentalsequences. The absence of molecular phylogenetic dataand conclusive ultrastructural data from Postgaardi pre-cludes us from determining whether this lineage is also amember of the clade of microaerophiles. Until these dataare reported and the phylogenetic position of Postgaardi isdemonstrated more rigorously, we concur with a previoustaxonomic treatment for Postgaardi that recognizes thislineage as incertae sedis within the Euglenozoa [3]. Assuch, we conclude that it is premature to recognize thetaxon Postgaardea and view it as a synonym for P. mariage-rensis.In light of the previous discussion, we propose the name"Symbiontida" for the clade of microaerobic or anaerobiceuglenozoans consisting of the most recent ancestor of C.aureus that also possessed rod-shaped epibiotic bacteria,reduced or absent mitochondrial cristae, tubular extru-somes and a nucleus with permanently condensed chro-matin. This novel subclade of euglenozoans is recognizedon the basis of SSU rDNA-based phylogenetic data of lin-eages from several different low-oxygen environments.Although the ultrastructural characteristics listed aboveare expected to be present in most, if not all, members ofthe Symbiontida (the ultrastructural and molecular phyl-ogeny of another lineage in this clade will be publishedshortly; Breglia, Yubuki, Hoppenrath and Leander, inpreparation), this remains to be demonstrated withimproved knowledge of euglenozoan diversity from bothultrastructural and molecular phylogenetic perspectives.Phylogenetic (apomorphy-based) diagnosisEuglenozoa Cavalier-Smith 1981Symbiontida taxon nov. Yubuki, Edgcomb, Bernhard &Leander, 2009ApomorphyRod-shaped epibiotic bacteria above superficial layer ofmitochondrion-derived organelles with reduced or absentcristae, homologous to the organization in Calkinsiaaureus, the type species (Figures 2, 4).Page 19 of 22(page number not for citation purposes)essarily reflect homology. Independently derived physicalrelationships between epibiotic bacteria and mitochon-BMC Microbiology 2009, 9:16 http://www.biomedcentral.com/1471-2180/9/16Extended diagnosis of the type speciesCalkinsia aureus Lackey, 1960, emend., Yubuki, Edgcomb,Bernhard & Leander, 2009Paraxonemal rods present in flagella; kinetoplast DNAand pellicle strips absent; long complex transitional zonebetween the basal bodies and the axonemes. Rod-shapedepibiotic bacteria on perforated orange extracellularmatrix. Cell with a large nucleus on the anterior ventralside and a battery of tubular extrusomes linked to anextrusomal pocket located adjacent to the nucleus. Feed-ing apparatus supported by both fibrous structures andmicrotubules that are derived from ventral root (VR).Small subunit ribosomal RNA gene sequence(EU753419) distinguishes Calkinsia aureus from all othersymbiontid species.ConclusionMolecular phylogenies inferred from SSU rDNA demon-strate that C. aureus is closely related to several marineenvironmental sequences collected from low-oxygenenvironments, forming a novel subgroup within theEuglenozoa, referred to here as the "Symbiontida".Improved understanding of these flagellates is necessaryfor further demonstrating the cellular identity of the Sym-biontida and for reconstructing the evolutionary radiationof the euglenozoan lineage. In this study, we characterizedthe detailed ultrastructure of C. aureus and demonstratedall of the euglenozoan synapomorphies (e.g. flagellarapparatus) and several cellular innovations associatedwith symbiotic interactions with epibiotic bacteria (e.g.,complex extracellular matrix). We also demonstratednovel ultrastructural systems found in this species, such asthe extrusomal pocket.Environmental sequencing surveys from different low-oxygen environments around the world suggest that manysymbiontid lineages have yet to be discovered and charac-terized. Continued exploration into the overall diversityof this group should contribute significantly to our under-standing of eukaryotic evolution, especially in low-oxygenenvironments.AbbreviationsAF: anterior flagellum; B: epibiotic bacteria; Ba: bacteriot-roph; CGS: congregated globule structure; DB: dorsalbasal body; DF: dorsal flagellum; DL: dorsal lamina; DR:dorsal root; E: extrusome(s); EP: extrusomal pocket; Eu:eukaryotroph; Ex: extracellular matrix; G: Golgi body; GL:glycocalyx; IF: intermediate fiber; IL: IR-associated lamina;IR: intermediate root; LF: left fiber; LM: light microscope;LMt: linked microtubules; MtD: mitochondrion-derivedorganelle; N: nucleus; Nu: nucleolus; Os: osmotroph;SBB: Santa Barbara Basin; SCB: sodium cacodylate buffer;SEM: scanning electron microscope; VB: ventral basalbody; T: tomentum; TEM: transmission electron micro-scope; VF: ventral flagellum; VR: ventral root.Authors' contributionsNY carried out all of the LM, SEM, TEM and molecularphylogenetic work, wrote the first draft of the paper andparticipated in the collection of sediment samples fromthe SBB. VPE and JMB, the Chief Scientist, coordinatedand funded the research cruise to the SBB. BSL funded andsupervised the collection and interpretation of theultrastructural and molecular phylogenetic data and con-tributed to writing the paper. All authors have read,edited, and approved the final manuscript.Additional materialAcknowledgementsThis work was supported by grants to BSL from the Tula Foundation (Cen-tre for Microbial Diversity and Evolution), the National Science and Engi-neering Research Council of Canada (NSERC 283091-04) and the Canadian Institute for Advanced Research, Program in Integrated Microbial Biodiver-sity. Funding for the collection of sediments and participation of VPE and JMB in this research was provided by the US National Science Foundation Additional file 1Maximum likelihood (ML) analysis of 29 taxa focusing on the posi-tion of Calkinsia aureus within the Euglenozoa clade. Two jakobids, Andalucia incarcerata and A. godoyi, are used as outgroups in this anal-ysis. The short environmental sequences are excluded from the dataset used in Figure 11 and fast-evolve euglenids sequences, Ploeotia, Menoidium and Astasia, are included. ML bootstrap values greater than 50% are shown. Thick branches indicate Bayesian posterior probabilities over 0.95. Ba, bacteriotroph; Eu, eukaryotroph; Os, osmotroph; Ph, pho-totroph. GenBank accession numbers of the sequences analyzed are shown in parentheses.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2180-9-16-S1.eps]Additional file 2Maximum likelihood (ML) analysis of 25 taxa focusing on the posi-tion of Calkinsia aureus within the Euglenozoa clade. Two jakobids, Andalucia incarcerata and A. godoyi, are used as outgroups in this anal-ysis. The short environmental sequences are removed from the dataset used in Figure 11 and fast-evolve euglenids sequences, Dinema, Ploeo-tia, Menoidium and Astasia, are excluded. ML bootstrap values greater than 50% are shown. Thick branches indicate Bayesian posterior proba-bilities over 0.95. Ba, bacteriotroph; Eu, eukaryotroph; Ph, phototroph. GenBank accession numbers of the sequences analyzed are shown in parentheses.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2180-9-16-S2.eps]Page 20 of 22(page number not for citation purposes)OSF: oblique striated fibrous structure; PF: posterior flag-ellum; Ph: phototroph; RF: right fiber; S: somatonema;grant MCB-060484. We also acknowledge the constructive feedback from four anonymous reviewers.BMC Microbiology 2009, 9:16 http://www.biomedcentral.com/1471-2180/9/16References1. Keeling PJ, Burger G, Durnford DG, Lang BF, Lee RW, Pearlman RE,Roger AJ, Gray MW: The tree of eukaryotes.  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