A photosynthetic alveolate closely relatedto apicomplexan parasitesRobert B. Moore1,2*, Miroslav Obornık3*, Jan Janouskovec3, Tomas Chrudimsky3, Marie Vancova3, David H. Green4,Simon W. Wright5, Noel W. Davies6, Christopher J. S. Bolch7, Kirsten Heimann8, Jan Slapeta9,Ove Hoegh-Guldberg10, John M. Logsdon Jr2 & Dee A. Carter1
Many parasitic Apicomplexa, such as Plasmodium falciparum, contain an unpigmented chloroplast remnant termed theapicoplast, which is a target for malaria treatment. However, no close relative of apicomplexans with a functionalphotosynthetic plastid has yet been described. Here we describe a newly cultured organism that has ultrastructural featurestypical for alveolates, is phylogenetically related to apicomplexans, and contains a photosynthetic plastid. The plastid issurrounded by four membranes, is pigmented by chlorophyll a, and uses the codon UGA to encode tryptophan in the psbAgene. This genetic feature has been found only in coccidian apicoplasts and various mitochondria. The UGA-Trp codon andphylogenies of plastid and nuclear ribosomal RNA genes indicate that the organism is the closest known photosyntheticrelative to apicomplexan parasites and that its plastid shares an origin with the apicoplasts. The discovery of this organismprovides a powerful model with which to study the evolution of parasitism in Apicomplexa.
Alveolata Cavalier-Smith, 1991 (emended by ref. 1)Chromerida phyl. nov.
Chromera velia gen. et sp. nov.
Etymology. Chromera (feminine), derived from the English wordschromophore and meront, because in pure culture the pigmentedplastid was inherited through cell division; velia (feminine), a mod-ern Italian proper name, meaning veiled or concealed (Supplemen-tary Information).Holotype/hapantotype. Z.6967 (Australian Museum, Sydney), pre-served culture embedded in PolyBed 812 (electron micrographsshown in Fig. 1a, b) and separately in absolute ethanol. The cultureis NQAIF136 (North Queensland Algal Culture and IdentificationFacility, James Cook University, Townsville, Australia). The clonalculture consists of dividing immotile organisms. Culture submissiondate, 25 February 2004; culture isolation date, 13 December 2001;isolator, R.B.M.Locality. Scleractinian coral Plesiastrea versipora (type host;Lamarck, 1816) (Metazoa: Cnidaria: Faviidae) obtained fromSydney Harbour, New South Wales, Australia (latitude 33u 509
38.7699 S; longitude 151u 169 4499 E) at ,3-m depth. Collection date,7 December 2001. Collectors: T. Starke-Peterkovic and L. Edwards.Referred material. Additional cultures are CCAP 1602/1 (CultureCollection of Algae and Protozoa, Scottish Association of MarineScience, UK) and CCMP2878 (Provasoli-Guillard Center forCulture of Marine Phytoplankton, Maine, USA).Diagnosis. Unicellular (Fig. 1a). The immotile life stage reproducesby binary division (Fig. 1b), but is not restricted to binary division.The immotile life stage is spherical to sub-spherical, 5–7mm in dia-meter in the G1 phase of the cell cycle. Cell diameter is up to 9.5 mm in
other cell cycle phases. A golden-brown, cone-shaped plastid is pre-sent. Immediately after completion of binary division, nascent cellscontain a single plastid only. Thylakoid lamellae are in stacks of threeor more (Fig. 1c). The plastid is bounded by four membranes (Fig. 1d)and contains chlorophyll a, but no other chlorophylls. A microporeis present (Fig. 1c). Internal cilium/cilia are present and anchoredat the cell periphery, extending to the periplastidal compartment(Fig. 1a, f, g). Cortical alveoli are flattened, with underlying micro-tubules (Fig. 1e). The position of attachment of internal ciliumto the cell periphery is defined as the apex of the immotile cell.There is a single, large mitochondrion ,1mm in diameter (Fig. 1a).Mitochondrial cristae are lamellar, ampulliform and tubular instructure. Vesicles of diameter ,1mm attach to and surround thelarge mitochondrion. The cell wall surface is smooth, with a raisedridge ,85 nm wide, extending around an incomplete circle andforking periodically (Supplementary Information). Chromera veliais free-living or associated with stony corals; it is the type speciesof the phylum Chromerida. Chromerida differ from all knownalveolates1 (Supplementary Information) in having a photosyntheticsecondary plastid bearing chlorophyll a, but no chlorophyll c2.
Plastids of alveolates and their medical significance
The alveolates are a major lineage of protists that are defined by thepossession of subsurface alveoli (flattened membrane-bound vesi-cles) supported by microtubules, as well as the presence of micro-pores and mitochondria with ampulliform or tubular cristae1,3.Alveolates are divided into three main phyla: the ciliates, apicom-plexans and dinoflagellates. The group Apicomplexa1 (Levine, 1970;emended by Adl et al. 2005 (ref. 1)) is composed mostly of parasitesthat are united by the possession of a set of secretory organelles
*These authors contributed equally to this work.
1School of Molecular and Microbial Biosciences, University of Sydney, Darlington, New South Wales 2006, Australia. 2Roy J. Carver Center for Comparative Genomics, Department ofBiological Sciences, University of Iowa, Iowa City, Iowa 52242-1324, USA. 3Biology Centre of the Academy of Sciences of the Czech Republic, Institute of Parasitology, and University ofSouth Bohemia, Faculty of Science, Branisovska 31, 37005 Ceske Budejovice, Czech Republic. 4Scottish Association for Marine Science, Dunstaffnage Marine Laboratory Oban, ArgyllPA37 1QA, UK. 5Australian Antarctic Division, Kingston, Tasmania 7050, Australia. 6Central Science Laboratory, University of Tasmania, Hobart, Tasmania 7001, Australia. 7School ofAquaculture, University of Tasmania, Launceston, Tasmania 7250, Australia. 8School of Marine and Tropical Biology, James Cook University, Townsville, Queensland 4811, Australia.9Faculty of Veterinary Science, University of Sydney, Camperdown, New South Wales 2006, Australia. 10Centre for Marine Studies, University of Queensland, St Lucia, Queensland4072, Australia.
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underlying an oral structure at the anterior apex of the cell4 (the‘apical complex’), and other characters. Within the phylum is amonophyletic subgroup of obligate parasites that comprises some6,000 taxa5. These present a major burden to livestock and humanhealth. Many contain a relic plastid termed the apicoplast6. Amongthe apicomplexans, it is specifically hemosporidians, piroplasms(both groups are blood parasites, including Plasmodium, that causemalaria) and coccidians (for example, Toxoplasma gondii7 and theveterinary pathogen Eimeria) that possess an apicoplast. Becauseanimals do not possess plastids, the apicoplast represents an oppor-tunity to target these parasites with treatments that are relativelyharmless to mammalian hosts8.
The reduced 35-kilobase genome and imported proteome of thePlasmodium apicoplast have been exhaustively studied. Severalcritical pathways are localized in the apicoplast, including fatty acidand isoprenoid biosynthesis6. However, not all apicomplexanspossess this organelle. No evidence of a plastid has been found inGregarina, the only gregarine examined so far9. Similarly, the water-borne parasite Cryptosporidium lacks an apicoplast10,11. Finally, thereis no published evidence for apicoplasts in colpodellids, a group ofnon-parasitic apicomplexans that possess an apical complex oforganelles used for predation on algal and protist prey4,12.
In the absence of an extant alga that represents the ancestral pho-tosynthetic state of these diverse apicomplexans, the evolutionarydescent of the apicoplast has instead been indicated by taxonomicand phylogenetic affiliation of apicomplexans to particular algae.Gene phylogenies relate the apicoplasts to the chloroplasts of a subsetof dinoflagellate algae—those that are pigmented by the chromo-phore peridinin13,14. It is thought that the common ancestor of peri-dinin dinoflagellates and apicomplexans possessed a chromalveolateplastid containing the specific combination of chlorophyll a andchlorophyll c13,15. Whereas peridinin dinoflagellates retained theplastid, degeneration of the photosynthetic chromalveolate plastidoccurred independently in many other dinoflagellates and alsooccurred independently in apicomplexans16. In a range of dinoflagel-lates, photosynthetic plastids were ingested and replaced the chromal-veolate plastid16. In other dinoflagellates, the chromalveolate plastidwas lost and not replaced, resulting in heterotrophy4,15,17. In parasiticapicomplexans the plastid remains, but in a non-photosynthetic form.Here we show that C. velia is a relative of parasitic apicomplexans andcolpodellids, and bears a photosynthetic plastid that is related mostclosely to apicoplasts but also to peridinin dinoflagellate plastids,affirming a common ancestry. Chromera velia can live independentlyof its host and is easily maintained in culture. As well as providing amodel to study apicomplexan evolution, we predict that C. velia willbe of practical use in high-throughput screening of prospective anti-apicoplast drugs.
Evolutionary position of Chromera velia
The three ultrastructural features diagnostic of alveolates1,3 are allpresent in C. velia: first, a micropore occurs in the cell periphery(Fig. 1c); second, subsurface alveoli are present and supported bymicrotubules (Fig. 1e); and third, the mitochondrion (Fig. 1a) con-tains ampulliform and tubular cristae (Supplementary Information).
Molecular phylogenetic analyses of nuclear genes showed thatC. velia is more closely related to apicomplexan parasites than tophotosynthetic dinoflagellates. In bayesian and maximum likelihoodanalyses of nuclear large subunit (LSU) rDNA sequences, C. veliabranched at the root of the Apicomplexa (Fig. 2a), with this positioncorroborated by a topology test (Fig. 2b) and by slow–fast analysis(Supplementary Information). Phylogeny of nuclear small subunit(SSU) rDNA sequences also supported a close relationship betweenC. velia and apicomplexans, with the new taxon branching at the rootof colpodellids (Fig. 2c). Although the position of Chromera on thistree had relatively low bootstrap and posterior probability support(maximum likelihood bootstrap 68, posterior probability 0.90),
cf
pl1
pl2pl2
n2
pl1
m
n1
cf
*
*
pl
n
plm
ic
iinv
ic
a b
mi
cwpl
c
mv
d
pc
ecw
fg
ic
plpl
g
ic1
bb1ic2
pl
pl2
pl1
cf
Figure 1 | Ultrastructure of new alveolate Chromera velia. a, Interphase (scalebar, 2mm). iinv, interphase invagination (black arrow); m, mitochondrion; n,nucleus; pl, plastid. b, Binary division. The apex of each daughter cell is markedby an asterisk. The mitochondrion of the mother cell is centrally positioned atthe terminator of the cleavage furrow, between the nuclei of daughter cells 1and 2 (scale bar, 2mm). cf, cleavage furrow. c, Micropore (mi) and thylakoidlamellae. The micropore is an invagination of the plasma membrane. Anassociated vesicle in the cytoplasm is indicated (mv, micropore-associatedvesicle). Thylakoid lamellae in the plastid are in sets of three (white arrows) ormore (scale bar, 200 nm). cw, cell wall. d, The two pairs of plastid membranesseparate at the periplastidal compartment (white triangles). The outer pairforms the periplastidal compartment (scale bar, 200 nm). pc, periplastidalcompartment. e, Alveoli and supporting microtubules. Alveoli lying beneaththe plasma membrane abut each other closely (at black arrows) and areunderlain by microtubules (white arrow) (scale bar, 500 nm). f, Maintenanceof the plastid (pl) in a cone shape is aided by one or more internal cilia (ic, whitetriangle) anchored at the apex of the cell (scale bar, 2mm). g, Magnification ofboxed area in f. Internal cilium ic1 extends inward from basal body bb1 (whitetriangle), which is attached to the cell periphery. ic1 and bb1 join at a terminalplate (black arrow). ic2 (white arrow) emerges perpendicular to ic1 (scale bar,500 nm). a, b, Hapantotype Z.6967 (Australian Museum, Sydney).
Figure 2 | Nuclear and plastid phylogenies of Chromera velia. a, Bayesianphylogenetic tree of nuclear LSU rDNA inferred from 2,740 characters(GenBank accession EU106870). b, Topology tests of the placement of the C.velia branch with respect to branches of the nuclear LSU rDNA tree.Numbered branches are indicated in a. Topology test results are: the P-valuefor the approximately unbiased test (au) calculated from the multiscalebootstrap; the non-parametric bootstrap probability calculated from themultiscale bootstrap (np); the bootstrap probability calculated in the non-multiscale manner (bp); the bayesian posterior probability calculated by thebayesian-information-criterion approximation (pp); and the P-values of theKishino–Hasegawa test (kh), the Shimodaira–Hasegawa test (sh), theweighted Kishino–Hasegawa test (skh) and the weighted
Shimodaira–Hasegawa test (wsh). c, Bayesian phylogenetic tree of nuclearSSU rDNA inferred from 1,285 characters (GenBank accession DQ174732).d, Topology tests of the placement of the C. velia branch with respect tobranches of the nuclear SSU rDNA tree. Numbered branches are indicated inc. e, Bayesian phylogenetic tree of plastid SSU rDNA gene sequences inferredfrom 811 characters (GenBank accession EU106871). f, Bayesianphylogenetic tree of the PsbA photosynthesis protein inferred from 319characters (GenBank accession EU106869). g, Topology tests of theplacement of the C. velia branch with respect to branches of the PsbA tree.Numbered branches are indicated in f. On all trees, black stars indicatebranches with bayesian posterior probabilities higher than 0.99 andmaximum likelihood bootstrap support higher than 90%.
topology tests rejected alternative placement of Chromera at theroot of dinozoans (Fig. 2d).
The lineage of the C. velia plastid was analysed using plastid rDNAand PsbA, a plastid protein that is part of photosystem II. In thephylogenetic analysis of plastid SSU rDNA, the C. velia chloroplastbranched at the root of the apicoplasts with good support (Fig. 2e). Itwas not possible to include sequences from the peridinin-pigmentedplastids of dinoflagellates in the plastid SSU rDNA analyses becausetheir high divergence caused their position in trees to be unstable.The PsbA sequence of C. velia was found to be conserved whencompared with that of other photosynthetic eukaryotes (Supplemen-tary Information), and phylogenetic analysis of PsbA was thereforerestricted to relevant taxa to avoid the effects of homoplasy acrossunrelated lineages. Taxa were selected based on the strong relation-ships shown between C. velia, dinoflagellates and stramenopiles onthe nuclear rDNA trees. The selected set included dinoflagellates,other chromalveolates and rhodophytes (Fig. 2f). Apicoplasts couldnot be included as they do not possess the psbA gene. Althoughmaximum likelihood support across the PsbA protein tree was lim-ited, there was significant bayesian support for known relationships,such as a grouping of stramenopile and dinoflagellate secondaryplastids (posterior probability 0.97, maximum likelihood 65), anda grouping of haptophyte secondary plastids as neighbour to thesetwo (posterior probability 0.99, maximum likelihood 90). The ana-lysis supported C. velia as a relative of peridinin dinoflagellate plas-tids (posterior probability 0.96, maximum likelihood 50). Topologytesting (Fig. 2g) corroborated the most likely placement of the C. veliaplastid as closest sister group to the plastids of peridinin dinoflagel-lates, as was expected given that apicomplexans were not included inthe analysis.
Unique features of the Chromera velia plastid
The psbA gene of C. velia contains an unusual codon that links theplastid to the apicoplast lineage. All other eukaryotic algae use UGGcodons to encode tryptophan at seven conserved positions in the
gene. The C. velia gene instead uses UGA codons at these positions(Supplementary Information). The UGA-Trp codon is unpreced-ented in any photosynthetic plastid and has only been found in theapicoplasts of coccidians and in various mitochondria6,18.
The C. velia plastid contains thylakoid lamellae in stacks of threeor more (Fig. 1c), resembling the arrangement in the plastids ofperidinin-pigmented dinoflagellates19. It displays novel pigmenta-tion, with chlorophyll a, violaxanthin and a novel carotenoid asthe major components, and b,b-carotene as a minor component(Supplementary Information). No other chlorophylls are present.Mass spectrometry analysis indicated that the novel carotenoid isan isomer of isofucoxanthin (Supplementary Information). Thisfinding is consistent with the Chromera plastid being red-derived,as isomers of isofucoxanthin are absent from plastids of the greenlineage2. Pulse amplitude modulation fluorescence analysis con-firmed that photosynthesis occurs in Chromera (SupplementaryInformation). Assuming that the apicomplexan–dinoflagellate groupwas ancestrally photosynthetic, the absence of chlorophyll c in C.velia was unexpected, as peridinin dinoflagellates normally possessthis pigment. We propose that secondary loss of chlorophyll c couldhave occurred in early apicomplexans.
An ultrastructural feature in common between the C. velia plastidand apicoplasts is the number of surrounding membranes. It isgenerally presumed that the number of membranes surrounding astable plastid can decrease but not increase during its evolutionaryspecialization20. The C. velia plastid is surrounded by four mem-branes (Fig. 1d). Reports vary in their estimates of the number ofmembranes surrounding apicomplexan plastids. Three-dimensionalreconstruction of the P. falciparum apicoplast indicated three mem-branes21, supplemented with additional inner and outer membranecomplexes. A similar reconstruction of the apicoplast of the cocci-dian T. gondii found spatial alternation of two and four membranes22.By comparison, the plastids of peridinin dinoflagellates are sur-rounded by three membranes19,20. We suggest that the four mem-branes bounding the C. velia plastid may represent the numbersurrounding the ancestor of apicoplasts and peridinin dinoflagellateplastids.
Concluding remarks
Phylogenetic analyses support the description of Chromera velia as analveolate, possessing a photosynthetic plastid that lies in the samesecondary endosymbiotic lineage as apicoplasts. The ultrastructureand photosynthetic pigment profile of C. velia are consistent with achromalveolate-affiliated ancestry. Figure 3 presents a model of theevolutionary history of C. velia, apicomplexans and dinoflagellatesbased on the phylogeny of the nuclear and plastid lineages and theretention or loss of plastid characteristics. Chromera velia representsthe closest known photosynthetic relative of apicomplexan parasites.
METHODS SUMMARYChromera velia was isolated from the stony coral Plesiastrea versipora (Faviidae)
from Sydney Harbour (Australia) by a variation of a procedure23 normally used
to isolate intracellular symbionts of the genus Symbiodinium (Supplementary
Information). Unicellular lines were generated by streaking raw colonies onto an
agar-gelled minimal growth medium, picking single colonies and regrowing in
liquid medium (Supplementary Information). Genomic DNA was extracted and
genes (nuclear SSU and LSU rDNA, and plastid SSU rDNA and psbA) wereamplified and sequenced. Purity of cultures was checked by sequencing multiple
nuclear SSU rDNA clones from each unialgal line (Supplementary Information).
Sequences were aligned, and phylogenetic analyses were performed using maxi-
mum likelihood and bayesian inference. Selected data sets were analysed using a
slow–fast method. Specimens for transmission electron microscopy (TEM) were
prepared using a freeze-substitution method24 and examined by TEM. Scanning
electron microscopy (SEM) specimens were prepared using standard methods
(Supplementary Information). Pigments were extracted and analysed by a com-
bination of high-performance liquid chromatography (HPLC), ultraviolet and
visible (UV/Vis ) spectra analysis and mass spectrometry (MS), and were iden-
tified by comparison of their retention times and spectra to those of mixed
standards obtained from known cultures (Supplementary Information).
Figure 3 | Evolution of Chromera velia, apicomplexans and dinoflagellates.The path in green traces the maintenance of photosynthesis. Characteristicsof the terminal nodes of coccidia, hemosporidians and piroplasms aregeneralized. The gregarine shown is Gregarina niphandroides9. TheCryptosporidium species represented is C. parvum11,25. ‘Other dinozoans’includes non-photosynthetic species: Perkinsus atlanticus (filled black circle,plastid present26) and Oxyrrhis marina (open circle, no plastid evident27).The dinozoan branch bearing ‘replaced plastids’ is a symbolic branchrepresenting many such branches that obtained tertiary plastidsindependently. Heterotrophic dinoflagellates have characters as for Oxyrrhismarina. The tree is a consensus of data presented in this paper and otherpublished relationships10,12,13,28–30.
Received 15 September 2007; accepted 9 January 2008.
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Supplementary Information is linked to the online version of the paper atwww.nature.com/nature.
Acknowledgements This work was supported by an ARC grant to D.A.C. andO.H.-G.; an ABRS grant to D.A.C.; grants from the Czech Science Foundation,Academy of Sciences of the Czech Republic and Czech Ministry of Education toM.O.; University of Iowa start-up funds and an NSF grant to J.M.L.; and a Universityof Tasmania Institutional Research grant to C.J.S.B. We thank A. McMinn for pulseamplitude modulation data, A. Simpson for analytical suggestions, R. Andersen forculture backup, and A. Polaszek and M. Garland for taxonomic opinions.
Author Contributions R.B.M. isolated the strain while in the D.A.C. laboratory, thenwhile in the J.M.L. laboratory designed the AToL SSU primers and the psbA primers,cloned and sequenced multiple copies of the SSU rRNA gene, a copy of the plastidSSU rRNA gene and initial sections of the psbA and LSU rRNA genes, then assignedprecedented methods for culture fixation, and wrote and finalized the draft of thepaper; M.O. led and performed phylogenetic analyses of the sequence data, clonedand sequenced a copy of the plastid SSU rDNA gene using different primers thanR.B.M., and co-wrote the draft of the paper; M.O. and M.V. performed the TEM andSEM data collection; J.J. and T.C. cloned and sequenced near full-length LSU rDNAand psbA genes and undertook extensive phylogenetic analysis; T.C. performedmito-red staining; S.W.W. and N.W.D. performed pigment analysis and interpretedpigment data; R.B.M., K.H., C.J.S.B. and J.S. interpreted TEM data and assignedtaxonomy; K.H. and R.B.M. performed light microscopy; R.B.M., M.O., T.C., K.H.,D.H.G. and C.J.S.B. maintained cultures. D.H.G. cloned and sequenced the LSUrRNA gene, using different PCR primers than T.C. and J.J.; R.B.M., M.O., D.H.G.,K.H., J.S., O.H.-G., J.M.L., C.J.S.B. and D.A.C. designed research, interpretedevolutionary, ecological and microbiological data, and performed extensive editingand revision.
Author Information Reprints and permissions information is available atwww.nature.com/reprints. Correspondence and requests for materials should beaddressed to D.A.C. ([email protected]).
A photosynthetic alveolate closely related toapicomplexan parasitesRobert B. Moore, Miroslav Obornık, Jan Janouskovec,Tomas Chrudimsky, Marie Vancova, David H. Green,Simon W. Wright, Noel W. Davies, Christopher J. S. Bolch,Kirsten Heimann, Jan Slapeta, Ove Hoegh-Guldberg,John M. Logsdon Jr & Dee A. Carter
Nature 451, 959–963 (2008)
In Fig. 2c of this Article, the GenBank accession number DQ174732was incorrectly cited. The correct accession number is DQ174731(Chromera velia).
ERRATUMdoi:10.1038/nature06872
Hax1-mediated processing of HtrA2 by Parlallows survival of lymphocytes and neuronsJyh-Rong Chao, Evan Parganas, Kelli Boyd, Cheol Yi Hong,Joseph T. Opferman & James N. Ihle
Nature 452, 98–102 (2008)
In Fig. 1a of this Letter, the light blue line was incorrectly labelledas Hax1/Bak DKO, n 5 13. The light blue line should be labelledHax1/Bax DKO, n 5 13.
ERRATUMdoi:10.1038/nature06898
Strong dispersive coupling of a high-finessecavity to a micromechanical membraneJ. D. Thompson, B. M. Zwickl, A. M. Jayich, Florian Marquardt,S. M. Girvin & J. G. E. Harris
Nature 452, 72–72 (2008)
In the print and HTML versions of this Letter, Fig. 2b was printedincorrectly. The PDF version published online is correct. The cor-rected Fig. 2b is shown below.
Membrane displacement (nm)1,2008004000
100
102
101
1.0
2.0
0.0
3.0
Lase
r d
etun
ing
(GH
z)
b
CORRECTIONS & AMENDMENTS NATUREjVol 452j17 April 2008
