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Morphostasis in a novel eukaryote illuminates the evolutionary transition from phagotrophy to phototrophy:… Yamaguchi, Aika; Yubuki, Naoji; Leander, Brian S Mar 8, 2012

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RESEARCH ARTICLE Open AccessMorphostasis in a novel eukaryote illuminates theevolutionary transition from phagotrophy tophototrophy: description of Rapaza viridis n. gen.et sp. (Euglenozoa, Euglenida)Aika Yamaguchi†, Naoji Yubuki† and Brian S Leander*†AbstractBackground: Morphostasis of traits in different species is necessary for reconstructing the evolutionary history ofcomplex characters. Studies that place these species into a molecular phylogenetic context test hypotheses aboutthe transitional stages that link divergent character states. For instance, the transition from a phagotrophic mode ofnutrition to a phototrophic lifestyle has occurred several times independently across the tree of eukaryotes; one ofthese events took place within the Euglenida, a large group of flagellates with diverse modes of nutrition.Phototrophic euglenids form a clade that is nested within lineages of phagotrophic euglenids and that originatedthrough a secondary endosymbiosis with green algae. Although it is clear that phototrophic euglenids evolvedfrom phagotrophic ancestors, the morphological disparity between species representing these different nutritionalmodes remains substantial.Results: We cultivated a novel marine euglenid, Rapaza viridis n. gen. et sp. ("green grasper”), and a green alga,Tetraselmis sp., from the same environment. Cells of R. viridis were comprehensively characterized with lightmicroscopy, SEM, TEM, and molecular phylogenetic analysis of small subunit rDNA sequences. Ultrastructural andbehavioral observations demonstrated that this isolate habitually consumes a specific strain of Tetraselmis prey cellsand possesses a functional chloroplast that is homologous with other phototrophic euglenids. A novel feedingapparatus consisting of a reduced rod of microtubules facilitated this first and only example of mixotrophy amongeuglenids. R. viridis also possessed a robust photoreception apparatus, two flagella of unequal length, euglenoidmovement, and a pellicle consisting of 16 strips and one (square-shaped) whorl of posterior strip reduction. Themolecular phylogenetic data demonstrated that R. viridis branches as the nearest sister lineage to phototrophiceuglenids.Conclusions: The unusual combination of features in R. viridis combined with its molecular phylogenetic positioncompletely conforms to the expected transitional stage that occurred during the early evolution of phototrophiceuglenids from phagotrophic ancestors. The marine mixotrophic mode of nutrition, the preference for green algalprey cells, the structure of the feeding apparatus, and the organization of the pellicle are outstanding examples ofmorphostasis that clarify pivotal stages in the evolutionary history of this diverse group of microbial eukaryotes.* Correspondence: bleander@mail.ubc.ca† Contributed equallyThe Department of Botany and Zoology, Beaty Biodiversity Research Centerand Museum, University of British Columbia, 6270 University Blvd.,Vancouver, British ColumbiaV6T 1Z4, CanadaYamaguchi et al. BMC Evolutionary Biology 2012, 12:29http://www.biomedcentral.com/1471-2148/12/29© 2012 Yamaguchi et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.BackgroundMorphostasis and transitional traitsThe reconstruction of early stages in the evolution of com-plex characters requires awareness of different degrees ofmorphostasis in character states as reflected in the unityand diversity of organisms. Evolutionary biologists whostudy groups of organisms with rich fossil records (e.g.,molluscs, vertebrates, diatoms and foraminiferans) havethe luxury of comparing both extinct and extant species toinfer patterns of character evolution. Evolutionary biolo-gists focused on microbial eukaryotes are most oftenrestricted to comparisons of character states present inextant species to infer patterns of character evolution [1].As expected, many species have retained traits that arecompletely compatible with those that are either inferredor observed to have been present in distant ancestors (e.g.,Nautilus, Latimeria, Acanthostega, Diarthrognathus andArchaeopteryx; [2-4]). The discovery, characterization, andmolecular phylogenetic analyses of species like these testhypotheses about trait evolution and illuminate the transi-tional stages that link divergent character states.Among the most significant events in the evolutionaryhistory of eukaryotes is the transition from a phago-trophic mode of life to a phototrophic mode of life,which has occurred several times independently [5-7].This switch in nutritional mode has profound effects onthe overall morphology and behavior of the descendantspecies. For instance, phagotrophic organisms tend to behighly motile in order to acquire food items and areusually composed of pliable cells or tissues that canaccommodate ingested material; phototrophic organ-isms, by contrast, tend to be non-motile, sessile orplanktonic and are usually composed of cells or tissuesencased in a rigid wall for protection or structural integ-rity. The evolutionary transition from phagotrophy tophototrophy is therefore non-trivial, and the intermedi-ate stages that bridge the morphological traits associatedwith these two lifestyles are important to understand.With this in mind, we have discovered and characterizeda novel marine species with a suite of character statesthat completely conforms to the inferred transitionalstage between the phagotrophic and phototrophic life-styles present in a diverse group of microbial eukaryotes,namely the Euglenida.A brief overview of euglenid biology and evolutionaryhistoryThe Euglenida is a large group of marine and freshwaterflagellates that contains more than 1,000 described spe-cies with diverse modes of nutrition, including phago-trophy (bacterivory and eukaryovory), osmotrophy(sensu stricto) and phototrophy [8]. The best synapo-morphy for this group of microbial eukaryotes is a cellsurface consisting of longitudinally or helically arrangedpellicle strips that articulate along their lateral margins[9]. Many species of phagotrophic, osmotrophic (sensustricto), and phototrophic euglenids are capable ofrhythmic cellular deformations called “euglenoid move-ment” (syn. “metaboly”) that is facilitated by adjacentstrips sliding past one another at their zones of articula-tion [8-13]. Most phagotrophic euglenids have a robustfeeding apparatus comprised of two main bundles ofmicrotubules called “rods” and four interior “vanes” thatare organized like a pinwheel in transverse section[9,14,15]. The feeding apparatus is highly developed inphagotrophic euglenids and highly reduced in osmo-trophic and phototrophic euglenids [16,17].Studies that have placed the morphological diversity ofeuglenids into a molecular phylogenetic context demon-strate that the mode of nutrition corresponds with gen-eral ultrastructural and behavioral traits [8,9]. Althoughit is expected that euglenid cells are capable of absorb-ing nutrients from the environment regardless of otherabilities to acquire nutrients (e.g., phagotrophy or photo-trophy), euglenids can be separated into four functionalgroups based on distinct nutritional modes: bacterivores(cells that usually feed on bacteria-relatively small parti-cles), eukaryovores (cells that usually feed on microbialeukaryotes-relatively large particles), primary osmo-trophs (cells that have lost a feeding apparatus and arelimited to the absorption of nutrients), and phototrophs(cells capable of acquiring nutrients through photo-synthesis). Bacterivores and eukaryovores are each para-phyletic, and the latter evolved from the former;primary osmotrophs and phototrophs are monophyletic,and each group evolved from eukaryovorous ancestorsindependently [8,9,16,18]. There are a couple of caveatsto these designations: (1) the bacterivorous and eukaryo-vorous designations are not immune from exceptions (e.g., some Petalomonas species are relatively large andcan consume yeast cells); (2) the loss of photosynthesiswithin phototrophic euglenids has led to several differ-ent lineages of “secondary” osmotrophs (cells that retaina non-photosynthetic plastid plus other ultrastructuralfeatures of phototrophic species; e.g., Euglena longa);and (3) the term “osmotrophy” refers to euglenid speciesthat are limited to the absorption of nutrients and is notintended to imply that photosynthetic and phagotrophiceuglenids are incapable of the absorbing nutrients fromthe environment.Nonetheless, bacterivorous euglenids (e.g., Petalomo-nas, Ploeotia and Entosiphon) glide along substratesusing one or two flagella, have a simple to complexfeeding apparatus, and tend to be smaller with a rigidpellicle consisting of relatively few longitudinal strips (i.e., 4-12). Eukaryovorous euglenids (Peranema, Hetero-nema and Urceolus) also glide along substrates usingone or two flagella, have a complex feeding apparatus,Yamaguchi et al. BMC Evolutionary Biology 2012, 12:29http://www.biomedcentral.com/1471-2148/12/29Page 2 of 16and tend to be larger with a flexible pellicle consistingof a relatively large number of helical strips (i.e., 16-60).The two main groups of euglenids that have lost phago-trophy, namely phototrophs and primary osmotrophs,contain lineages capable of euglenoid movement andseveral derived lineages that have lost euglenoid move-ment. The presence of euglenoid movement in somephototrophs and primary osmotrophs is inferred toreflect morphostasis from their eukaryovorous ancestors[8,9,13,18]. Primary osmotrophic euglenids swim in thewater column using one or two flagella, lack a feedingapparatus, and have either a flexible or a fused pellicleconsisting of a relatively large number of helical strips(i.e., usually more than 16-30, Rhabdomonas costata has6-8). Phototrophic euglenids are separated into twomain groups: the Eutreptiales and the Euglenales. Mem-bers of the Eutreptiales are largely marine, swim in thewater column using two or more flagella, contain chlor-oplasts, lack a conspicuous feeding apparatus, possess apronounced photoreception apparatus, and have a flex-ible pellicle consisting of a relative large number of heli-cal strips (i.e.,16-50). Members of the Euglenales arelargely freshwater, normally swim in the water columnusing one emergent flagellum, contain chloroplasts, lacka conspicuous feeding apparatus, possess a pronouncedphotoreception apparatus, and have a flexible or rigidpellicle consisting of a relatively large number of(usually) helical strips (i.e., 16-120). The pellicle ofphototrophic euglenids can be distinguished from allother euglenids by the presence of “posterior whorls ofstrip reduction” (i.e., patterns of strips that terminatebefore reaching the posterior end of the cell). The actualnumber of pellicle strips and posterior whorls of stripreduction present on any particular cell reflects phyloge-netic relationships and fundamental developmentalmechanisms in euglenids [8-10,13,19].The chloroplasts of phototrophic euglenids originatedthrough a secondary endosymbiotic relationship betweeneukaryovorous euglenids and green algae. The pellicleand feeding apparatus in species like Heteronema andUrceolus best approximate the inferred features presentin the most recent non-photosynthetic ancestor ofphototrophic euglenids [8,13,16]; the chloroplasts of pra-sinophyceans like Pyramimonas best approximate theinferred features present in the most recent ancestor ofall euglenid chloroplasts [20-22]. Nonetheless, there arestill substantial differences between the morphologicaland behavioral features of eukaryovorous euglenids andphototrophic euglenids that limit our ability to fully por-tray the evolutionary transition between these two verydifferent lifestyles. An improved understanding of eugle-nid diversity is expected to help bridge remaining gapsin our knowledge of this key event. In this vein, we havediscovered and comprehensively characterized a novelmarine euglenid with a uniquely “mixotrophic” lifestyle;this species contains a functional chloroplast and habi-tually consumes a specific strain of green algae. In thiscontext, the term “mixotrophy” refers to a euglenid thatis capable of both phagotrophy and phototrophy. Beha-vioral and ultrastructural data derived from high resolu-tion light microscopy, scanning and transmissionelectron microscopy (SEM and TEM), and molecularphylogenetic analysis of small subunit (SSU) rDNAsequences demonstrate that this novel species conformsto the expected transitional stage between phototrophiceuglenids and eukaryovorous ancestors.MethodsCollection of organism and maintenance of culturesSeawater samples were collected from a tide pool atPachena Beach, Bamfield, British Columbia, Canada (48°47.551’ N, 125° 06.974’ W) on June 18, 2010. The sam-ples were inoculated in Provasoli’s Enriched Seawater(PES) medium [23] and maintained at room temperaturefor one week. Tetraselmis sp. was isolated from theenrichment culture by micropipetting into sterile PESmedium. Rapaza viridis n. gen. et sp. was also isolatedfrom the enrichment culture into PES medium contain-ing Tetralselmis sp. as a food source. Both cultures wereincubated at 20°C under an illumination of 55-59 μmolphotons/m2/s with 12:12 light:dark (L:D) light regime.The cultures were transferred every 10 to 14 days bybringing 1 ml of the culture into 25 ml of PES mediumand prey cells. The cultures of R. viridis n. gen. et sp.and Tetraselmis sp. have been deposited into the Ameri-can Type Culture Collection (ATCC), Manasas, VA,USA as PRA-360 and PRA-361, respectively.Light microscopyDifferential interference contrast (DIC) light micro-graphs were generated using a Zeiss Axioplan 2 imagingmicroscope equipped with Leica DC500 digital camera.Digital videos of live cells were generated using a ZeissAxioplan 2 equipped with a Q imaging Microimager IIdigital camera and Q Capture v 2.8.1 software.Growth experimentsCells of R. viridis were exposed to several different foodsources in addition to Tetraselmis sp. ATCC PRA-361.Cultures of Tetraselmis sp. NEPCC365, Tetraselmis sp.NEPCC498, T. striata NEPCC487 and T. tetrathelleNEPCC 483 were obtained from the Canadian Centerfor the Culture of Microorganisms (CCCM) at the Uni-versity of British Columbia, Vancouver, BC, Canada. Aculture of Navicula sp. ATCC PRA-314 was obtainedfrom the American Type Culture Collection (ManasasVA, USA). Cultures of the prasinophyte Pycnococcus sp.,the chlorophyte Dunaliella sp., and the dinoflagellateYamaguchi et al. BMC Evolutionary Biology 2012, 12:29http://www.biomedcentral.com/1471-2148/12/29Page 3 of 16Scrippsiella trochoidea were established in the lab fromdifferent marine environments independently.Cultures of R. viridis were starved of prey cells andobserved every 2-3 days until the death of all R. viridiscells was confirmed. We confirmed the existence of atleast one chloroplast in the starved R. viridis cells usinga Zeiss Axioplan 2 imaging microscope after one week,three weeks and five weeks from the start of starvation.Cultures of R. viridis were also grown in the dark at20°C. Prey cells (Tetraselmis sp., ATCC PRA-361) wereadded every 1-2 days until the death of all R. viridiscells was confirmed. As a control experiment, culturesgrown under normal light conditions were also treatedexactly like the cultures grown in the dark (e.g., thesame volume of prey cells was added at the same inter-vals of time).Scanning electron microscopyA culture of R. viridis was mixed in 1% (v/v) OsO4 inseawater at room temperature. The fixed cells weremounted on polycarbonate Millipore filters (13 mmdiam., 5 μm pore size) or glass plates coated with poly-L-lysine at room temperature for 15 min. The cells wererinsed with distilled water and dehydrated with a gradedethanol series from 30% to absolute ethanol beforebeing critical point dried with CO2 using a TousimisCritical Point Dryer. The dried cells were then coatedwith gold using a Cressington 208HR High ResolutionSputter Coater and observed with a Hitachi S-4700 fieldemission SEM.Transmission electron microscopyTwo different cultures were examined with transmissionelectron microscopy (TEM): (1) a culture starved of Tet-raselmis sp. cells for three weeks and (2) a culture thatwas fed cells of Tetraselmis sp. one hour prior to fixa-tion. The cultures were pre-fixed in 2.5% (v/v) glutaral-dehyde with 0.2 M sucrose in 0.1 M sodium cacodylatebuffer (SCB) (pH 7.2) at room temperature for 2 hours.The pre-fixed cells were washed in 0.2 M SCB (pH 7.2)twice and post-fixed in 1% (w/v) osmium tetroxide in0.2 M SCB (pH 7.2) at room temperature for 1 hour.The fixed cells were dehydrated through a graded seriesof ethanol and 100% acetone. The dehydrated cells werethen infiltrated with acetone-Epon 812 resin mixturesand 100% Epon 812 resin. Ultra-thin serial sections werecollected on copper Formvar-coated slot grids, stainedwith 2% (w/v) uranyl acetate and lead citrate, andobserved using a Hitachi H7600 TEM.DNA extraction, PCR amplification, alignment andphylogenetic analysisGenomic DNA was extracted using the MasterPureComplete DNA and RNA purification Kit (Epicentre,WI, USA) from a culture of R. viridis that was starvedof prey cells for three weeks. Polymerase chain reactions(PCR) were performed using PuRe Taq Ready-To-GoPCR beads kit (GE Healthcare, Buckinghamshire, UK).The nearly complete eukaryotic SSU rDNA gene wasamplified using the eukaryotic universal primers 5’-TGATCCTTCTGCAGGTTCACCTAC-3’ and 5’-GCGCTACCTGGTTGATCCTGCCAGT-3’ with thesame PCR protocol described by Breglia et al. (2010)[24]. The amplified DNA fragments were purified fromagarose gels using UltraClean 15 DNA Purification Kit(MO Bio, CA, USA), and subsequently cloned into theTOPO TA Cloning Kit (Invitrogen, CA, USA). Oneclone was sequenced using ABI Big-Dye reaction mix(BigDye 3.1) using the vector forward and reverse pri-mers and also internal primers (nomet1134R: 5’-TTTAAGTTTCAGCCTTGCG-3’ and SR4Eug: 5’-ACTGGAGGGCAAGYCTGGT-3’) oriented in bothdirections. The new sequence was initially identified byBLAST analysis, confirmed with molecular phylogeneticanalysis, and deposited into GenBank: AB679269.The SSU rRNA sequence from R. viridis was added toa 39-taxon alignment focused on the diversity of eugle-nids using representative kinetoplastids and diplonemidsas an outgroup. Ambiguously aligned positions and gapswere excluded from alignment, leaving 805 unambigu-ously aligned positions; the alignment is available fromthe authors upon request.Maximum likelihood (ML) analysis was performed onthe 39-taxon alignment using PAUP* version 4.0b10[25]. Prior to starting the ML analysis, we used Akaikeinformation criterion (AIC) test as implemented in thesoftware jModelTest 0.1.1. [26] to find the model of evo-lution that best fits the data set for the Maximum likeli-hood (ML) analysis. The result indicated that theTIM1ef, which allows for equal base frequencies andfour substitution rates (AC = GT; AT = CG; AG, CT)[27,28], plus gamma model should be used for this dataset. The parameters were as follows: assumed nucleotidefrequencies are equal; substitution rate matrix with A-Csubstitutions = 1.0000, A-G = 2.7002, A-T = 0.7141, C-G = 0.7141, C-T = 3.8573, G-T = 1.0000; proportion ofsites assumed to be invariable = 0 and rates for variablesites assumed to follow a gamma distribution withshape parameter = 0.3930. The ML tree was implemen-ted using the heuristic search option with TBR branchswapping. Bootstrap analyses [29] were carried out forML with 500 replicates to evaluate statistical reliability.The alignment was also analyzed with Bayesian meth-ods using the MrBayes 3.1.2 [30]. The program was setto operate the GTR model with a gamma distributionand four Monte-Carlo-Markov chains (MCMC) startingfrom a random tree. A total of 500,000 generations werecalculated with trees sampled every 100 generations.Yamaguchi et al. BMC Evolutionary Biology 2012, 12:29http://www.biomedcentral.com/1471-2148/12/29Page 4 of 16The first 1,250 trees in each run were discarded asburn-in using the sumt command. Posterior probabil-ities correspond to the frequency at which a given nodewas found in the post burn-in trees.Sequence availabilityThe SSU rDNA nucleotide sequences included in 39-taxon analyses for this paper are available from the Gen-Bank database under the following accession numbers:Anisonema acinus [GenBank:AF403160], Bihospitesbacati [GenBank:HM004354], Bodo saltans [GenBank:AY998648], Calkinsia aureus [GenBank:EU753419],Colacium sp. [GenBank:DQ140154], Dimastigellamimosa [GenBank:DQ207576], Dinema sulcatum [Gen-Bank:AY061998], Diplonema ambulator [GenBank:AF380996], Diplonema papillatum [GenBank:AF119811], Discoplastis spathirhyncha [GenBank:AJ532454], Distigma proteus [GenBank:AF106036],Entosiphon sp. [GenBank:AY425008], Entosiphon sulca-tum [GenBank:AF220826], Euglena gracilis [GenBank:AF283308], Euglena longa (as Astasia longa) [GenBank:AF112871], Euglena quartana (as Khawkinea quartana)[GenBank:U84732], Euglena stellata [GenBank:AF081590], Euglena viridis [GenBank:AF445460],Eutreptia viridis [GenBank:AF157312], Eutreptiella gym-nastica [GenBank:AF081590], Eutreptiella pomquetensis[GenBank:AJ532398], Ichthyobodo necator [GenBank:AY224691], Lepocinclis buetschlii [GenBank:AF096993],Menoidium cultellus [GenBank:AF295019], Monomor-phina sp. [GenBank:DQ140130], Neobodo designis [Gen-Bank:AF209856], Notosoleus ostium [GenBank:AF403159], Peranema sp. [GenBank:AY048919], Pera-nema trichophorum [GenBank:AF386636], Petalomonascantuscygni [GenBank:AF386635], Phacus aenigmaticus[GenBank:AF283313], Ploteotia costata [GenBank:AF525486], Rapaza viridis [GenBank: AB679269], Rhab-domonas costata [GenBank:AF295021], Rhynchomonasnasuta [GenBank:AY998642], Rhynchopus sp. [GenBank:AF380997], Strombomonas triquetra [GenBank:DQ140153], Trypanosoma evansi [GenBank:AY904050],Trypanosoma sp. [GenBank:EF375883].ArchivingA digital archive of this paper is available from PubMedCentral and print copies are available from libraries inthe following 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.).ResultsGeneral morphologyThe relaxed, (well-fed) swimming cells of Rapaza viridis n.gen. et sp. were oval, 10.5-38.2 μm long (mean ± SD =19.5 ± 7.3 μm, n = 50), and 2.9-15.1 μm wide (mean ± SD= 8.4 ± 3.3 μm, n = 50). The cells of R. viridis usuallyswam in a spiral pattern with the flagella moving rapidlyand chaotically. Euglenoid movement was also observedwhen the cells were feeding on Tetraselmis or when thecells were pressured by a cover glass (Figure 1A, Addi-tional file 1). Two flagella emerged from the flagellarpocket, were unequal in length, were adorned with hairs,and contained heteromorphic paraxial rods (Figures 1E,2A and 3B). The longer anterior flagellum was about 1.25times the length of the cell in the relaxed state and alwaysdirected forward. The shorter posterior flagellum wasabout 0.65 times the length of the relaxed cell and directedbackward; this flagellum was sometimes directed forwardand moved oar-like (Additional file 1). A robust anteriorstigma that was independent of the chloroplasts was com-prised of one to more than ten particles ranging from 0.8-1.3 μm in diam. (Figures 4B and 4D). A paraflagellar swel-ling formed a dense, lens-like body that was positionedwithin the flagellar pocket and against the stigma (Figure4D). The cytoplasm contained several ellipsoid paramylongrains (Figure 1B), large Golgi bodies, and mitochondriawith discoidal (paddle-shaped) cristae (Figure 4A). Thetypical euglenid nucleus contained permanently con-densed chromosomes and a large central nucleolus (Figure3A). The pellicle consisted of 16 helically arranged strips(Figures 4A and 5A) with delicate S-shaped frames thatwere supported by underlying microtubules (Figure 4C).Eight pellicle strips terminated before reaching the poster-ior end of the cell; these strips were organized in pairs thatformed the four corners of a square-like pattern when theterminating strips were traced with lines (Figure 2D). Thefeeding apparatus consisted of a feeding pocket and oneadjacent feeding rod comprised of 20 microtubules orga-nized in four rows (4-6-6-4) (Figures 3B, C and 3D). Wedid not observe any vanes or amorphous material asso-ciated with these microtubules (Figure 3).Feeding behaviorR. viridis ingested Tetraselmis cells when plentiful in theculture and were distinctly larger and bright green inthe presence of food (Figures 1G and 1H). The Tetrasel-mis cells, including their chloroplasts, were completelydigested over the course of about 12 hours. Whenstarved of Tetraselmis prey, the cells of R. viridis becamesmaller and colorless, except for the retention of at leastone intact chloroplast within the cytoplasm (Figures 1EYamaguchi et al. BMC Evolutionary Biology 2012, 12:29http://www.biomedcentral.com/1471-2148/12/29Page 5 of 16and 1F). R. viridis could not survive for more than 35days without exposure to a specific strain of Tetraselmissp. (PRA-361). Different strains of Tetraselmis spp. andseveral other species of microalgae were added to thecultures in an attempt to grow R. viridis on an alternatefood source: the prasinophytes Tetraselmis sp.NEPCC365, Tetraselmis sp. NEPCC498, T. striataNEPCC487, T. tetrathelle NEPCC 483, Pycnococcus sp.,the diatom Navicula sp. ATCC PRA-314, the chloro-phyte Dunaliella sp., and the dinoflagellate Scrippsiellatrochoidea. R. viridis rejected all of these prey choices.Adding Tetraselmis sp. (PRA-361) into a culture ofstarved R. viridis cells triggered a feeding frenzy. R. viri-dis captured Tetraselmis sp. (PRA-361) with the anteriorpart of the cell and either rotated rapidly or swam back-ward in a spiral pattern while dragging the prey cell(Figures 1C and 3A). Euglenoid movement was promi-nent during the processes of ingestion (Figures 1D, 2Cand Additional file 2). R. viridis was capable of comple-tely engulfing prey cells. In the process of engulfment,the theca (cell wall) of Tetraselmis was gradually loo-sened and peeled away by the repetitive peristalticFigure 1 Differential interference contrast (DIC) light micrographs (LM) of Rapaza viridis n. gen. et sp. showing general morphologicalfeatures of the species. A. Time series of a live cell showing euglenoid movement (arrow, stigma). B. LM showing paramylon grains(arrowheads) free in the cytoplasm. C. LM showing R. viridis capturing a Tetraselmis cell (arrowhead) with the anterior part of the cell. D. LMshowing R. viridis engulfing a Tetraselmis cell. The arrow points to the theca of Tetraselmis, and the arrowheads denote the expanded cytostomeof R. viridis. E-F. LMs of R. viridis cells that have been starved for one week (arrow, stigma; double arrowhead, euglenid chloroplast). G-H. LMs ofR. viridis cells that were fed 24 hours earlier. E-H. same magnification. Scale bars 10 μm in A, C-H; 5 μm in B.Yamaguchi et al. BMC Evolutionary Biology 2012, 12:29http://www.biomedcentral.com/1471-2148/12/29Page 6 of 16actions of euglenoid movement (Figures 1D and 2B-C).However, Tetraselmis cells with an intact theca couldalso be engulfed, but the theca was discharged from theanterior end of R. viridis cells soon afterwards. The con-tinuous actions of euglenoid movement facilitated thecomplete ingestion and posterior transport of anengulfed Tetraselmis cell (Additional file 2). The entireprocess of engulfment took 5-40 minutes; at this point,the R. viridis cell would slow down euglenoid move-ment, elongate, and begin to swim again. Cells of R. viri-dis exposed to an ample food supply contained severalingested cells of Tetraselmis at one time.The chloroplasts of Rapaza viridis and Tetraselmis spThere were two ultrastructurally distinct types ofchloroplasts present in well-fed cells of R. viridis: (1)chloroplasts corresponding to the ultrastructure ofgreen algae (i.e., Tetraselmis) and (2) chloroplastscorresponding to the ultrastructure of phototrophiceuglenids. The euglenid-type chloroplasts were sur-rounded by three membranes, contained thylakoids instacks of three, and contained one to three darklystained pyrenoids without associated paramylon gran-ules (Figures 5A, B and 5D). Double stacks of thyla-koids also penetrated the pyrenoids in the euglenid-type chloroplasts (Figures 5A and 5D). Electron-densestructures surrounded by a single membrane wereoften observed just outside the euglenid chloroplast(Figure 5C). The Tetraselmis-type chloroplasts wereoften discernible within the cytoplasm of R. viridis bylight microscopy because of their intraplastidial eye-spots. The Tetraselmis-type chloroplasts were also dis-tinguishable from the euglenid-type chloroplasts withTEM. A complete membrane surrounded engulfed Tet-raselmis cells (Figure 5F). The pyrenoids in the Tetra-selmis-type chloroplasts were surrounded by starchFigure 2 Scanning electron micrographs (SEM) of Rapaza viridis n. gen. et sp. A. SEM showing a relaxed elongated cell with helical pelliclestrips and two flagella. The anterior flagellum (AF) and the posterior flagellum (PF) insert into the anterior part of the cell. B. SEM showing an R.viridis cell that has bored a hole into the cell surface of a captured Tetraselmis cell (arrowhead). The internal contents of the captured Tetraselmiscell (arrow) are being withdrawn by myzocytosis. C. SEM showing R. viridis engulfing a Tetraselmis cell (arrowhead). D. SEM of R. viridis showingposterior strip reduction (*), which connected by lines, forms a square-shaped whorl. Scale bars 5 μm in A-C; 2 μm in D.Yamaguchi et al. BMC Evolutionary Biology 2012, 12:29http://www.biomedcentral.com/1471-2148/12/29Page 7 of 16Figure 3 Transmission electron micrographs (TEM) of Rapaza viridis n. gen. et sp. showing details of the feeding apparatus. A.Longitudinal TEM showing R. viridis capturing a Tetraselmis cell (arrow). The arrowhead indicates the theca (cell wall) of Tetraselmis, which isbeginning to unravel. The chloroplast of R. viridis (Rcp) and the nucleus (N) containing permanently condensed chromosomes and a largenucleolus (Nu) is also visible. B. Transverse TEM through the flagellar pocket (flp) showing paraxial rods (PR), the pocket (fep) and themicrotubules of the feeding rod (arrow). C. Transverse TEM through the feeding rod showing 20 microtubules organized in four rows (4-6-6-4).D. Longitudinal TEM through the feeding apparatus showing the feeding pocket (’fep’ and arrows) and microtubules of the rod (arrowheads).Scale bars 5 μm in A; 1 μm in B and D; 200 nm in C.Yamaguchi et al. BMC Evolutionary Biology 2012, 12:29http://www.biomedcentral.com/1471-2148/12/29Page 8 of 16grains and did not contain penetrating thylakoids (Fig-ure 5E). Two membranes, rather than three, sur-rounded the Tetraselmis-type chloroplasts (Figure 5F).Moreover, unlike the euglenid-type chloroplasts, intra-plastidial eyespot globules were easily seen at the per-iphery of Tetraselmis-type chloroplasts (Figures 5Dand 5G).Starved cells of R. viridis almost always contained atleast one intact chloroplast (Figures 1E and 1F). TEMdemonstrated that this enduring chloroplast had theeuglenid-type ultrastructure (Figure 5A). When cells ofR. viridis were grown in the absence of light, they couldnot survive for more than one week. The result of thisdark growth experiment was consistent even in the pre-sence of an ample and continuous supply of Tetraselmisprey cells. Cells of R. viridis required both photosynth-esis and (Tetraselmis) prey cells in order to survive.Molecular phylogenetic positionWe determined the nearly complete sequence of theSSU rRNA gene of R. viridis (2,669 bp). Molecular phy-logenetic analysis of the 39-taxon alignment demon-strated that phototrophic euglenids (syn. euglenophytes),including secondary osmotrophic species (Euglena longaand E. quartana), formed a robust monophyletic group(100% bootstrap value and 1.00 Bayesian posterior prob-ability) (Figure 6). R. virdis formed the nearest sisterlineage to clade of phototrophic euglenids with relativelyhigh statistical support (80% bootstrap value and 1.00Bayesian posterior probability).Figure 4 Transmission electron micrographs (TEM) of Rapaza viridis n. gen. et sp. showing details of the cytoplasm, pellicle andphotoreception apparatus. A. Transverse TEM showing the mitochondrion (M-arrowhead), the Golgi body (G-arrowhead) and 16 pellicle strips(arrows). B. Transverse TEM showing the stigma (arrows) and the paraflagellar swelling (Pa). C. Transverse TEM showing the S-shaped frames ofthe pellicle strips (arrowheads) and underlying microtubules (arrows). D. Sagittal TEM showing the stigma (arrows) and the paraflagellar swelling(Pa). Scale bars 1 μm in A, B and D; 500 nm in C.Yamaguchi et al. BMC Evolutionary Biology 2012, 12:29http://www.biomedcentral.com/1471-2148/12/29Page 9 of 16Figure 5 Transmission electron micrographs (TEM) of Rapaza viridis n. gen. et sp. showing details of two types of chloroplasts. A.Transverse TEM showing the chloroplast of R. viridis and 16 pellicle strips (arrowheads). Pairs of thylakoid membranes (arrows) penetrate thepyrenoids (Py). The black arrow indicates the the pellicle strip that is shown in Figure 5B. B. Higher magnification TEM of Figure 5A focusing onthe three membranes that surround the chloroplast of R. viridis (arrowheads) and the associated stacks of thylakoids (Ty). The black arrowindicates the the pellicle strip that is shown in Figure 5A. C. High magnification TEM showing electron-dense structures surrounded by a singlemembrane (arrowheads) that are situated near a chloroplast. D. TEM showing two different types of chloroplasts: The chloroplast of Tetraselmissp. (Tcp) contains an eyespot (arrowheads); the chloroplast of R. viridis (Rcp) does not. E. Longitudinal TEM showing an engulfed Tetraselmis cell,including the nucleus (N), pyrenoid (Py) and associated starch (S) and chloroplast (Cp). Arrowheads indicate the pellicle strips of R. viridis. F. Highmagnification TEM showing the food vacuole of R. viridis (R-arrow) and the cell membrane of the engulfed Tetraselmis cell (T-arrow). Whitearrowheads mark the two membranes that surround the chloroplast of Tetraselmis sp. Black arrowheads mark the pellicle strips of R. viridis. G.High magnification TEM showing the eyespot (arrows) within the chlorplasts of Tetraselmis sp. Scale bars 1 μm in A, C and G; 200 nm in B andF; 4 μm in D and E.Yamaguchi et al. BMC Evolutionary Biology 2012, 12:29http://www.biomedcentral.com/1471-2148/12/29Page 10 of 16DiscussionAlong with all of the expected euglenid features (e.g.,pellicle strips, mitochondria with discoidal cristae, anucleus with permanently condensed chromosomes, andparaxial rods), R. viridis possessed two heterodynamicflagella (rather than one emergent flagellum), swimmingbehavior, and euglenoid movement that was mostreminiscent of members within the Eutreptiales. Likemost species of Eutreptia and Eutreptiella, R. viridislives in marine environments, which stands in contrastto the freshwater lifestyles of the vast majority of specieswithin the Euglenales (e.g., Euglena, Phacus, Lepocinclisand Trachelomonas). However, R. viridis is clearly dis-tinct from members of the Eutreptiales because of itsFigure 6 Phylogenetic position of Rapaza viridis n. gen. et sp. within the Euglenozoa as inferred from SSU rRNA gene sequences.Maximum-likelihood (ML) tree constructed using TIM1ef + G model of evolution on an alignment of 39 taxa and 805 unambiguously alignedsites, including 298 constant positions and 432 informative positions, using seven kinetoplastid and three diplonemid sequences as theoutgroup. ML bootstrap values greater than 50% are shown. Thick branches indicate Bayesian posterior probabilities (PP) over 0.95. The branchleading to the fast-evolving Entosiphon clade has been shortened by one half (indicated by ‘1/2’).Yamaguchi et al. BMC Evolutionary Biology 2012, 12:29http://www.biomedcentral.com/1471-2148/12/29Page 11 of 16eukaryovorous behavior and associated feeding appara-tus. Moreover, the molecular phylogenetic analyses ofSSU rDNA sequences supports the placement of R. viri-dis as the nearest sister lineage to all photosyntheticeuglenids (i.e., the clade consisting of the Eutreptialesand the Euglenales) rather than sister to or within theEutreptiales. R. viridis is also clearly distinct from othereukaryovorous euglenids in having features present inall other phototrophic euglenids, such as functionalchloroplasts and a robust plastid-independent photore-ception apparatus (i.e., a stigma plus a photosensoryswelling at the base of the dorsal flagellum). Althoughthere is inconclusive evidence that the eukaryovoreUrceolus cyclostomus might also possess a weakly devel-oped photoreception apparatus [16], R. viridis representsthe first indisputable example of a eukaryovorous eugle-nid with photosensory ability. This would enable R. viri-dis to maintain its position in the water column inorder to increase the likelihood of encountering its pre-ferred photosynthetic prey cells (Tetraselmis sp.). How-ever, because R. viridis is also photosynthetic, thephotosensory ability could have more to do with main-taining an optimal position in the light for its ownphotosynthesis. Overall, the molecular phylogeneticposition and combination of morphological and beha-vioral traits in R. viridis is more than just novel; thesedata completely conform to the inferred ancestral traitsthat existed during the evolutionary transition fromeukaryovorous lifestyles to phototrophic lifestyles.Transitional character statesThe molecular phylogenetic analysis of SSU rDNAplaced R. viridis squarely between phagotrophic andphototrophic euglenids as the nearest sister lineage tothe entire euglenophyte clade (i.e., all phototrophiceuglenids and their secondary osmotrophic descen-dants). This phylogenetic context alone makes R. viridisa particularly intriguing candidate for gaining newinsights into euglenid character evolution, especially inregard to the endosymbiotic origin of chloroplasts. Theultrastructural and behavioral features in R. viridis wereperhaps even more compelling because they were sointermediary between the features already described inphagotrophic and phototrophic euglenids (Figure 7); thisis especially relevant for characters associated withmodes of nutrition, the feeding apparatus, the flagellarapparatus and the pellicle.MixotrophyR. viridis was unable to survive when placed in the darkor when deprived of Tetraselmis prey cells; therefore, R.viridis required both photosynthesis and phagotrophy tosatisfy its overall nutritional requirements. This “mixo-trophic” mode of nutrition is rare and has only beendescribed in a few groups of eukaryotes, such as insome dinoflagellates, haptophytes and stramenopiles[31-33]. Until now, mixotrophy was completelyunknown within the Euglenida. Nonetheless, a mixo-trophic mode of nutrition is a logical transitional stageduring the establishment of endosymbiotic partnershipsbetween (ancestrally) phagotrophic host cells and photo-trophic prey cells (Figure 7). Once the required horizon-tal gene transfer events and gene retargetingmechanisms established a permanent photosyntheticorganelle within a new host cell, the ability to lose pha-gotrophy and to rely exclusively on phototrophybecomes possible [34,35]. This has clearly happened inseveral different lineages of eukaryotes independently,including in the most recent ancestor of all photo-trophic euglenids [8]. Therefore, the mixotrophic modeof nutrition in R. viridis is entirely consistent with themolecular phylogenetic position of this species and clari-fies an important transitional stage in the origin ofphototrophy in euglenids (Figure 7).Feeding apparatusThe main components of the feeding apparatus in mostphagotrophic euglenids are two robust rods that canextend the entire length of the cell. Phototrophic eugle-nids have retained a highly reduced feeding apparatuswithout rods [9,17]. A few phototrophic euglenidswithin the Eutreptiales, however, have retained smallclusters of microtubules that are inferred to be homolo-gous to the rods of phagotrophic euglenids [36,37]. Thefeeding rod we characterized in R. viridis is intermediatein size and organization between the robust rods in pha-gotrophic species and the reduced rods in the Eutrep-tiales (e.g., Eutreptiella eupharyngea) (Figure 7). Thisresult is entirely consistent with the molecular phyloge-netic position of this species, substantiates the abovehomology statement, and clarifies an important transi-tional stage in the origin of phototrophy in euglenids(Figure 7).Flagellar apparatusPhagotrophic euglenids generally glide along surfaceswith two relatively thick heterodynamic flagella: theanterior (dorsal) flagellum is directed forward andtwitches at the tip; the posterior (ventral) flagellum isdirected backwards and glides against the substrate [8].Phototrophic (and osmotrophic) euglenids generallyswim in the water column using one or two flagella thatmove somewhat erratically or move in a controlled fig-ure-eight configuration that pulls the cell forward.These modes of swimming allow phototrophic euglenidsto exploit the water column above the substrate [9]. Thecells of R. viridis were capable of swimming in the watercolumn in a pattern that was most reminiscent of spe-cies within the Eutreptiales. Therefore, R. viridis repre-sents the first example of a eukaryovorous euglenid thatis also capable of swimming in the water column; this isYamaguchi et al. BMC Evolutionary Biology 2012, 12:29http://www.biomedcentral.com/1471-2148/12/29Page 12 of 16another feature that is consistent with the molecularphylogenetic position of this species and that clarifies animportant transitional stage in the origin of phototrophyin euglenids (Figure 7).PellicleThe substructure, number, and organization of pelliclestrips reflects phylogenetic relationships within euglenids[8-11,13,19]. For instance, members of the Euglenalestend to have strips with relatively thick proteinaceousframes and underlying projections that interconnectadjacent strips [9,11,13,38]. Phagotrophic species, (plas-tic) primary osmotrophic species, and members of theEutreptiales have relatively thin proteinaceous frameswithout projections. The strip substructure of R. viridiswas most similar to the strips in members of theEutreptiales and eukaryovores like Dinema and Pera-nema, which is consistent with the transitional stateexpected between eukaryovorous and phototrophicFigure 7 Illustrations showing the transitional character states present in Rapaza viridis n. gen. et sp. that link phagotrophic euglenidswith phototrophic euglenids. Illustrations of five different euglend cells emphasizing the size of the feeding apparatus, the number of flagella,and the presence or absence of chloroplasts, pyrenoids, paramylon grains and a stigma. The illustrations below each representative cell show thenumber of pellicle strips and whorls of posterior strip reduction as indicated by different colors. Pellicle strips that reach the posterior end of thecell are shown in yellow; strips that terminate before reaching the posterior end are shown in blue and purple. The solid grey lines that connectthe terminating strips represent each exponential whorl of reduction. Note that the cell representing the Eutreptiales has one zigzag(exponential) whorl associated with two staggered subwhorls. The illustrations are based on the ultrastructural studies of Ploeotia [45-47],Heteronema scaphurum [48], the Eutreptiales (e.g., Eutreptia pertyi) [14,19,36,49], and the Euglenales (e.g., Euglena laciniata for the pattern ofpellicle strips and Eugelna gracilis for general shield-shaped plastids) [19,50,51].Yamaguchi et al. BMC Evolutionary Biology 2012, 12:29http://www.biomedcentral.com/1471-2148/12/29Page 13 of 16euglenids [11,13]. The total number of strips in R. viri-dis (16) was relatively low in comparison to Peranemaand Eutreptia and more consistent with the number ofstrips found in eukaryovores like Dinema and primaryosmotrophs like Distigma (16-22) [10,16].Posterior whorls of strip reduction have only beenobserved in phototrophic euglenids; however, some rela-tively obscure patterns of posterior strip reduction havealso been observed in one eukaryovore (Peranema; [13]).The number and pattern of posterior whorls of stripreduction varies in different species and reflects phyloge-netic relationships within phototrophic euglenids [19,39].Posterior whorls of strip reduction are usually “exponen-tial” (i.e., the number of strips that terminate on a whorlequals the number of strips that pass through the whorl,leading to a pattern whereby the number of strips ishalved at each whorl), but in some species the whorls areseparated into two or more “linear” subwhorls (i.e., thenumber of strips that terminate is the same for all sub-whorls; Figure 7) [10,13,39]. The range for the number ofexponential whorls of strip reduction known so far in theEuglenales is two to four [19]. One member of theEutreptiales (Eutreptia pertyi) has been shown to possesstwo linear subwhorls of posterior strip reduction thatcorresponds to one exponential whorl of strip reduction[13] (Figure 7). Aside from a few derived species of Pha-cus [40], R. viridis is the first and only euglenid describedso far with one clear exponential whorl of strip reduction;this precisely corresponds to the expected character statethat was present in the most recent ancestor of all eugle-nids possessing posterior whorls of strip reduction (i.e.,all phototrophic species) [9,13,19]. The eight strips thatterminate on the whorl of strip reduction in R. viridisform four distinct pairs that, when connected by lines,define the equidistant corners of a square (Figure 2D).This pattern of posterior strip reduction is novel amongall euglenids characterized so far.The chloroplasts of Rapaza viridisSome dinoflagellates, animals, and foraminiferans pos-sess “kleptochloroplasts”, which are transiently func-tional chloroplasts derived from algal food. The hostcells retains these chloroplasts for a short period of timein order to obtain products of photosynthesis as asource of nutrition; hosts are not capable of maintainingchloroplasts as permanent organelles that can be inher-ited from generation to generation. Kleptochloroplastsare ultimately digested, and the host organism needs toreplenish them by regularly consuming algal food.Eukaryovorous euglenids (e.g., Peranema and Urceolus)do not have chloroplasts but they often engulf microal-gae as their primary source of food, so well-fed cells cansuperficially appear full of chloroplasts; these chloro-plasts disappear when the eukaryovorous cell is starved.By contrast, the cells of R. viridis always retained atleast one chloroplast even when starved for over amonth and cannot survive in the dark even when regu-larly exposed to an abundant food supply. Therefore,cells of R. viridis require both photosynthesis and (Tet-raselmis) prey cells in order to survive.Our ultrastructural observations of well-fed andstarved cells of R. viridis demonstrated that this speciesusually contains both stable chloroplasts and transientTetraselmis chloroplasts that can be readily distin-guished from one another. The stable chloroplasts in R.viridis were surrounded by three membranes, lacked anintraplastidial eyespot and contained pyrenoids pene-trated by thylakoid membranes (Figures 5A, B and 5D).These features are consistent with the ultrastructure ofthe chloroplasts in many species of phototrophic eugle-nids, such as Colacium cyclopicolum and Euglena deses[41,42]. However, the presence of more than one pyre-noid per chloroplast in some cells was a novel feature ofR. viridis that has not been observed in any other spe-cies of euglenophyte so far (Figures 3A and 5A).Nonetheless, euglenid chloroplasts arose as a conse-quence of an endosymbiotic relationship between a eukar-yovorous euglenid and green algal prey cells that weremost similar to prasinophyceans [9,20-22]. The fact thatour isolate requires both photosynthesis and (Tetraselmis)prey cells in order to survive provides additional supportfor this inference. However, the relatively close relation-ship between the stable, euglenid-type chloroplasts andthe transient Tetraselmis chloroplasts in R. viridis mightcreate additional challenges when characterizing each ofthem at the genomic level. Nonetheless, the molecularphylogenetic position of R. viridis suggests that the gen-ome of their stable, euglenid-type chloroplasts will offersome of the most compelling insights into the earliest evo-lutionary stages in the endosymbiotic acquisition of chlor-oplasts in euglenids and beyond.ConclusionThe unusual combination of features in R. viridis com-bined with its molecular phylogenetic position comple-tely conforms to the expected transitional stage thatoccurred during the early evolution of phototrophiceuglenids from phagotrophic ancestors. The marine mix-otrophic mode of nutrition, the preference for green algalprey cells, the structure of the feeding apparatus, and theorganization of the pellicle are outstanding examples ofmorphostasis that clarify pivotal stages in the evolution-ary history of this diverse group of microbial eukaryotes.Formal taxonomic descriptionsEuglenozoa [43]Euglenida [44] (ICZN)Rapaza n. gen. Yamaguchi, Yubuki & Leander 2012Yamaguchi et al. BMC Evolutionary Biology 2012, 12:29http://www.biomedcentral.com/1471-2148/12/29Page 14 of 16DescriptionCells solitary and mixotrophic. Two heterodynamic fla-gella, unequal in length. Euglenoid movement with heli-cally arranged pellicle strips. A minimum of onediscoidal chloroplast surrounded by three membranesand with pyrenoids penetrated by double stacks of thyla-koids. Robust extra-plastidic stigma and paraflagellarswelling. Eukaryovorous on microalgae using a feedingapparatus consisting of one rod of microtubules, a feed-ing pocket, and no vanes.Type speciesRapaza viridis Yamaguchi, Yubuki & Leander 2012EtymologyLatin “rapax”, meaning seizing and grasping in referenceto the feeding behavior of the cell. The adjective ‘rapax’is used as substantive noun. Feminine.Rapaza viridis n. sp. Yamaguchi, Yubuki & Leander2012DescriptionSwimming cells are slender with a tapered posterior end,10.5-38.2 μm long (average, 19.5 μm), 2.9-15.1 μm wide(average, 8.4 μm); with two heterodynamic flagella,unequal in length, same in thickness; the longer flagel-lum about twice the length of the shorter flagellum;with stigma comprised of several (1 to more than 10)pigmented particles at the anterior part of the cell; ellip-soid paramylon grains free in the cytoplasm; pellicleconsisting of 16 helical strips; one square-shaped whorlof exponential strip reduction; feeding apparatus com-prised of a feeding rod consisting of four rows of 20microtubules (4-6-6-4); preferred prey Tetraselmis sp.Holotype and hapantotypesBoth 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; accession number MI-PR113) at theUniversity of British Columbia, Vancouver, Canada. Allfigures in the manuscript are based on the authenticculture, and Figure 1A has been selected as theholotype.DNA sequenceA sequence of the small subunit rRNA gene is depositedas GenBank Accession No. AB679269).Type localityPachena Beach, Bamfield, British Columbia, Canada (48°47.551’ N, 125° 06.974’ W), June 18, 2010.HabitatTide pools, marine.Authentic culturePRA-360. This culture is maintained in the AmericanType Culture Collection (ATCC), Manasas VA, USA.EtymologyThe specific epithet, viridis (green), refers to the color ofthe stable chloroplasts and prey cells. The binomial isLatin for “green grasper”.Additional materialAdditional file 1: A movie showing euglenoid movement andflagellar motility in Rapaza viridis n. gen. et sp.Additional file 2: A movie showing Rapaza viridis n. gen. et sp.engulfing a cell of Tetraselmis sp. (1.7 MB MOV). This movie is threetimes faster than actual speed.AbbreviationsAF: anterior flagellum; fep: feeding pocket; flp: flagellar pocket; G: Golgibody; LM: light microscope; M: mitochondrion; N: nucleus; Nu: nucleolus; Pa:paraflagellar swelling; PES: Provasoli’s Enriched Seawater; PF: posteriorflagellum; PR: paraxial rod; Rcp: chloroplast of R. viridis; Py: pyrenoid; S: starchgrain; SCB: sodium cacodylate buffer; SEM: scanning electron microscope;Tcp: chloroplast of Tetraselmis sp.; TEM: transmission electron microscope; Ty:thylakoids in stacks.AcknowledgementsThis research was supported by grants from the Tula Foundation (Centre forMicrobial Diversity and Evolution), the National Science and EngineeringResearch Council of Canada (NSERC 283091-09), the National ScienceFoundation (Assembling the Tree of Life NSF #EF-0629624), and theCanadian Institute for Advanced Research, Program in Integrated MicrobialBiodiversity. We also thank Erick James for access and assistance with thedigital movies. We also thank the UBC Bioimaging Facility for technicalassistance with the SEM.Authors’ contributionsAY and NY collected the water samples from Pachena Beach, BritishColumbia; established and maintained cultures of Rapaza virids andTetraselmis sp.; generated the LM, SEM, TEM and SSU rDNA sequence data;and drafted an initial version of the manuscript. BSL funded, facilitated, andsupervised the collection, interpretation and presentation of the behavioral,ultrastructural, and molecular phylogenetic data and wrote subsequentversions of the manuscript. All authors have read, edited and approved thefinal manuscript.Received: 15 November 2011 Accepted: 8 March 2012Published: 8 March 2012References1. Leander BS, Keeling PJ: Morphostasis in alveolate evolution. Trends EcolEvol 2003, 18:395-402.2. Johanson Z, Long JA, Talent JA, Janvier P, Warren JW: Oldest coelacanth,from the Early Devonian of Australia. Biol Lett 2006, 2:443-446.3. 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