A photosynthetic alveolate closely related to apicomplexan parasites

Article A photosynthetic alveolate closely related to apicomplexan parasites Robert B. Moore*1,2, Miroslav Oborník*3, Jan Janouškovec3, Tomáš Chrudimský3, Marie Vancová3, David H. Green4, Simon W. Wright5, Noel W. Davies6, Christopher J.S. Bolch7, Kirsten Heimann8, Jan Šlapeta9, Ove Hoegh-Guldberg10, John M. Logsdon Jr2, Dee A. Carter1. 1

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School of Molecular and Microbial Biosciences, University of Sydney, Australia. Roy J. Carver Center for 3 Comparative Genomics, Department of Biological Sciences, University of Iowa, USA. Biology Centre 4 ASCR, Institute of Parasitology, and University of South Bohemia, Czech Republic. Scottish Association for 5 6 Marine Science, UK. Australian Antarctic Division, Australia. Central Science Laboratory, University of 7 8 Tasmania, Australia. School of Aquaculture, University of Tasmania, Australia. James Cook University, 9 10 Australia. Faculty of Veterinary Science, University of Sydney, Australia. Centre for Marine Studies, University of Queensland, Australia. * These authors contributed equally

Abstract: Many parasitic Apicomplexa, such as Plasmodium falciparum, contain an unpigmented chloroplast remnant termed the apicoplast, which is a target for malaria treatment. However, no close relative of apicomplexans has yet been described with a functional photosynthetic plastid. Here we describe a newly cultured organism that has ultrastructural features typical for alveolates, is phylogenetically related to apicomplexans, and contains a photosynthetic plastid. The plastid is surrounded by four membranes, is pigmented by chlorophyll a, and uses the codon UGA to encode tryptophan in the PsbA gene. This genetic feature has been found only in coccidian apicoplasts and various mitochondria. The UGA-Trp codon and phylogenies of plastid and nuclear rRNA genes indicate that the organism is the closest known photosynthetic relative to apicomplexan parasites and that its plastid shares an origin with the apicoplasts. The discovery of this organism provides a powerful model to study the evolution of parasitism in Apicomplexa.

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Alveolata (Cavalier-Smith, 1991 emend. Adl et al. 20051) Chromerida phyl. nov. Chromera velia gen. et sp. nov. Etymology: Chromera (f.), derived from the English words chromophore and meront, because in pure culture the pigmented plastid was inherited through cell division; velia (f.), a modern Italian proper name, meaning veiled or concealed (Supplementary Information). Holotype / hapantotype: Z.6967 (Australian Museum, Sydney) preserved culture embedded in PolyBed 812 (electron micrographs shown in Fig. 1a, Fig. 1b) and separately in absolute ethanol. The culture is NQAIF136 (North Queensland Algal Culture and Identification Facility, James Cook University, Townsville, Australia). The clonal culture consists of dividing immotile organisms. Culture submission date: February 25th 2004. Culture isolation date: December 13th 2001. Isolator: R. Moore. Type host and locality: Scleractinian coral Plesiastrea versipora (Lamarck, 1816) (Metazoa: Cnidaria: Faviidae) obtained from Sydney Harbour, New South Wales, Australia (Latitude: 33° 50' 38.76" S, Longitude: 151° 16' 44" E) at ~3 m depth. Collection date: December 7th 2001. Collectors: Thomas Starke-Peterkovic and Les Murray. Additional cultures: CCAP 1602/1 (Culture Collection of Algae and Protozoa (Scottish Association of Marine Science, UK) and CCMP2878 (Culture Collection of Marine Protozoa, Maine, USA). Diagnosis. Unicellular (Fig. 1a). Immotile lifestage reproduces by binary division (Fig. 1b), but not restricted to binary division. Immotile lifestage spherical to sub-spherical, 5-7 µm in diameter in the G1 phase of the cell cycle. Diameter up to 9.5 µm in other cell cycle phases. Golden-brown cone shaped plastid present. Immediately after completion of binary division, nascent cells contain a single plastid only. Thylakoid lamellae in stacks of three or more (Fig. 1c). Plastid bounded by four membranes (Fig. 1d) and containing chlorophyll a, but no other chlorophylls. Micropore present (Fig. 1c). Internal cilium/cilia present and anchored at the cell periphery, extending to the periplastidal compartment (Fig. 1a, Fig. 1f, Fig. 1g). Cortical alveoli flattened, with underlying microtubules (Fig. 1e). Position of attachment of internal cilium to the cell periphery defined as the apex of the immotile cell. Single large mitochondrion ~1 µm diameter (Fig. 1a). Mitochondrial cristae lamellar, ampulliform and tubular. Vesicles of diameter ~1 µm attach to and surround the large 2

mitochondrion. Cell wall surface smooth with a raised ridge ~85 nm wide, extending around an incomplete circle and forking periodically (Supplementary information). Freeliving or associated with stony corals. Chromera velia is the type species of the phylum Chromerida. Chromerida differ from all known alveolates1 (Supplementary information) in having a photosynthetic secondary 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 the possession of subsurface alveoli (flattened membrane bound vesicles) supported by microtubules, as well as the presence of micropores, and mitochondria with ampulliform or tubular cristae1,3. Alveolates are divided into three main phyla: the ciliates, apicomplexans and dinoflagellates. The group Apicomplexa1 (Levine 1970, emend. Adl et al. 2005) is composed mostly of parasites, that are united by the possession of a set of secretory organelles underlying an oral structure at the anterior apex of the cell4 (the ‘apical complex’), and other characters. Within the phylum is a monophyletic subgroup of obligate parasites that comprises some 6000 taxa5. These present a major burden to livestock and human health. Many contain a relic plastid termed the apicoplast6. Among the apicomplexans, it is specifically hemosporidians and piroplasms, (blood parasites including Plasmodium, which causes malaria), and coccidians, (for example Toxoplasma gondii7 and the veterinary pathogen Eimeria) that possess an apicoplast. Since animals do not possess plastids, the apicoplast represents an opportunity to target these parasites with treatments that are relatively harmless to mammalian hosts8. The reduced 35 kb genome and imported proteome of the Plasmodium apicoplast have been exhaustively studied. Several critical pathways are localized in the apicoplast, including fatty acid and isoprenoid biosynthesis6. However, not all apicomplexans possess this organelle. No evidence of a plastid has been found in Gregarina, the only gregarine examined to date9. Likewise, the waterborne parasite Cryptosporidium lacks an apicoplast10,11. Finally, there is no published evidence for apicoplasts in colpodellids, a group of non-parasitic apicomplexans that possess an apical complex of organelles used for predation on algal and protist prey4,12.

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In the absence of an extant alga that represents the ancestral photosynthetic state of these diverse apicomplexans, the evolutionary descent of the apicoplast has instead been indicated by taxonomic and phylogenetic affiliation of apicomplexans to particular algae. Gene phylogenies relate the apicoplasts to the chloroplasts of a subset of dinoflagellate algae, those that are pigmented by the chromophore peridinin13,14. It is thought that the common ancestor of peridinin dinoflagellates and apicomplexans possessed a chromalveolate plastid containing the specific combination of chlorophyll a and chlorophyll c13,15. While peridinin dinoflagellates retained the plastid, degeneration of the photosynthetic chromalveolate plastid occurred independently in many other dinoflagellates and also occurred independently in apicomplexans16. In a range of dinoflagellates, photosynthetic plastids were ingested and replaced the chromalveolate plastid16. In other dinoflagellates the chromalveolate plastid was lost and not replaced, resulting in heterotrophy4,15,17.

In

parasitic

apicomplexans

the

plastid

remains,

but

in

a

nonphotosynthetic form. Here we show that C. velia is a relative of parasitic apicomplexans and colpodellids, and bears a photosynthetic plastid that is related most closely to apicoplasts but also to peridinin dinoflagellate plastids, affirming a common ancestry. Chromera velia can live independently of its host and is easily maintained in culture. As well as providing a model to study apicomplexan evolution, we predict C. velia will be 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 all present in C. velia: first, a micropore occurs in the cell periphery (Fig. 1c); second, subsurface alveoli are present supported by microtubules (Fig. 1e); and third, the mitochondrion (Fig. 1a) contains ampulliform and tubular cristae (Supplementary information). Molecular phylogenetic analyses of nuclear genes showed that C. velia is more closely related to apicomplexan parasites than to photosynthetic dinoflagellates. In Bayesian and maximum likelihood (ML) analysis of nuclear LSU rDNA sequences, C. velia branched at the root of the Apicomplexa (Fig. 2a), with this position corroborated by a topology test (Fig. 2b) and by slow-fast analysis (Supplementary information). Phylogeny of nuclear SSU rDNA sequences also supported a close relationship between C. velia and 4

apicomplexans, with the new taxon branching at the root of colpodellids (Fig. 2c). Although the position of Chromera on this tree had relatively low bootstrap and posterior probability support (ML bootstrap 68, posterior probability 0.90), topology tests rejected alternative placement of Chromera either as sister to all apicomplexans, or at the root of dinoflagellates and apicomplexans (Fig. 2d). The lineage of the C. velia plastid was analysed using plastid rDNA and PsbA, a plastid protein that is part of photosystem II. In the phylogenetic analysis of plastid SSU rDNA, the C. velia chloroplast branched at the root of the apicoplasts with good support (Fig. 2e). It was not possible to include sequences from the peridinin-pigmented plastids of dinoflagellates in the plastid SSU rDNA analyses because their high divergence caused their position in trees to be unstable. The PsbA sequence of C. velia was found to be conserved when compared to that of other photosynthetic eukaryotes (Supplementary information), and phylogenetic analysis of PsbA was therefore restricted to relevant taxa to avoid the effects of homoplasy across unrelated lineages. Taxa were selected based on the strong relationships shown between C. velia, dinoflagellates and stramenopiles on the nuclear rDNA trees. The selected set included dinoflagellates, other chromalveolates, and rhodophytes (Fig. 2f). Apicoplasts could not be included as they do not possess the psbA gene. Although ML support across the PsbA protein tree was limited, there was significant Bayesian support for known relationships, such as a grouping of stramenopile and dinoflagellate secondary plastids (posterior probability 0.97, ML 65), and a grouping of haptophyte secondary plastids as neighbour to these two (posterior probability 0.99, ML 90). The analysis supported C. velia as a relative of peridinin dinoflagellate plastids (posterior probability 0.96, ML 50). Topology testing (Fig. 2g), corroborated the most likely placement of the C. velia plastid as closest sister to the plastids of peridinin dinoflagellates, as was expected given that apicomplexans were not included in the analysis. Unique features of the Chromera velia plastid The psbA gene of C. velia contains an unusual codon that links the plastid to the apicoplast lineage. All other eukaryotic algae use UGG codons to encode tryptophan at six conserved positions in the gene. The C. velia gene instead uses UGA codons at these positions 5

(Supplementary information). The UGA-Trp codon is unprecedented in any photosynthetic plastid and has only been found in the apicoplasts of coccidians, and in various mitochondria6,18. The C. velia plastid contains thylakoid lamellae in stacks of three or more (Fig. 1c), resembling the arrangement in the plastids of peridinin-pigmented dinoflagellates19. It displays novel pigmentation, with chlorophyll a, violaxanthin, and a novel carotenoid as the major components, and β,β-carotene as a minor component (Supplementary information). No other chlorophylls are present. Mass spectrometry analysis indicated that the novel carotenoid is an isomer of isofucoxanthin (Supplementary information). This finding is consistent with the Chromera plastid being red-derived, as isomers of isofucoxanthin are absent from plastids of the green lineage2. Pulse amplitude modulation (PAM) fluorescence analysis confirmed that photosynthesis occurs in Chromera (Supplementary information). Assuming the apicomplexan-dinoflagellate group was ancestrally photosynthetic, the absence of chlorophyll c in C. velia was unexpected, as peridinin dinoflagellates normally possess this pigment. We propose that secondary loss of chlorophyll c could have occurred in early apicomplexans. An ultrastructural feature in common between the C. velia plastid and apicoplasts is the number of surrounding membranes. It is generally presumed that the number of membranes surrounding a stable plastid can decrease but not increase during its evolutionary specialisation20. The C. velia plastid is surrounded by four membranes (Fig. 1d). Reports vary in their estimates of the number of membranes surrounding apicomplexan plastids. Three-dimensional reconstruction of the P. falciparum apicoplast indicated three membranes21, supplemented with additional inner and outer membrane complexes. A similar reconstruction of the apicoplast of the coccidian T. gondii, found spatial alternation of two and four membranes22. By comparison, the plastids of peridinin dinoflagellates are surrounded by three membranes19,20. We suggest that the four membranes bounding the C. velia plastid may represent the number surrounding the ancestor of apicoplasts and peridinin dinoflagellate plastids. Summary 6

Phylogenetic analyses support the description of Chromera velia as an alveolate, possessing a photosynthetic plastid that lies in the same secondary endosymbiotic lineage as apicoplasts. The ultrastructure and photosynthetic pigment profile of C. velia are consistent with a chromalveolate-affliated ancestry. Figure 3 presents a model of the evolutionary history of C. velia, apicomplexans and dinoflagellates based on the phylogeny of the nuclear and plastid lineages and the retention or loss of plastid characteristics. Chromera velia represents the closest known photosynthetic relative of apicomplexan parasites. Methods summary Chromera 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) were amplified 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 maximum likelihood and Bayesian inference. Selected datasets 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 analyzed by a combination of HPLC/UV and Vis/MS and were identified by comparison of their retention times and spectra to those of mixed standards obtained from known cultures (Supplementary information).

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Figure legends Figure 1 | Ultrastructure of new alveolate Chromera velia. a, Interphase (bar 2 µm). b, Binary division. The apex of each daughter cell is marked by an asterisk. The mitochondrion of the mother cell is centrally positioned at the terminator of the cleavage furrow, between the nuclei of daughter cells 1 and 2 (bar 2 µm). c, Micropore and thylakoid lamellae.The micropore is an invagination of the plasma membrane. An associated vesicle in the cytoplasm is indicated. Thylakoid lamellae in the plastid are in sets of three (white arrows) or more (bar 200 nm). d, The two pairs of plastid membranes separate at the periplastidal compartment (white triangles). The outer pair forms the periplastidal compartment (bar 200 nm). e, Alveoli and supporting microtubules. Alveoli lying beneath the plasma membrane abut each other closely (at black arrows) and are underlain by microtubules (white arrow) (bar 500 nm). f, Maintenance of the plastid (pl) in a cone shape, is aided by one or more internal cilia (ic) anchored at the apex of the cell (bar 2 µm). g, Magnification of a. Internal cilium ic1 extends inward from basal body bb1 (white triangle) which is attached to the cell periphery. Ic1 and bb1 join at a terminal plate (black arrow). Ic2 emerges perpendicular to ic1 (bar 500 nm). a-b, Hapantotype Z.6967 (Australian Museum, Sydney). Abbreviations: n, nucleus; pl, plastid; m, mitochondrion; ic, internal cilia/cilium anchoring the plastid (white arrows); cf, cleavage furrow; cw, cell wall; iinv, interphase invagination (black arrow), bb, basal body; mi, micropore; mv, micropore-associated vesicle; pc, periplastidal compartment Figure 2 | Nuclear and plastid phylogenies of Chromera velia. a, Bayesian phylogenetic tree of nuclear LSU rDNA inferred from 2740 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 indicated in a. c, Bayesian phylogenetic tree of nuclear SSU rDNA inferred from 1285 characters, (Genbank accession DQ174732). d, Topology tests of the placement of the C. velia branch with respect to branches of the nuclear SSU rDNA tree. Numbered branches indicated in c. e, Bayesian phylogenetic tree of plastid SSU rDNA gene sequences inferred from 811 characters (Genbank accession EU106871). f, Bayesian phylogenetic tree of the psbA photosynthesis protein inferred from 319 characters (Genbank accession EU106869). g, Topology tests of the placement of the C. velia branch with respect to branches of the psbA tree. Numbered branches indicated in f. On all trees, black stars indicate branches with Bayesian posterior probabilities higher than 0.99 and ML bootstrap support higher than 90%. Figure 3 | Evolution of Chromera velia, apicomplexans and dinoflagellates. The path in green traces the maintenance of photosynthesis. Characteristics of the terminal nodes of coccidia, hemosporidians, and piroplasms are generalised. The gregarine shown is Gregarina niphandroides9. The Cryptosporidium species represented is C. parvum11,25. ‘Other dinozoans‘ includes non photosynthetic species: Perkinsus atlanticus (•, plastid present26), and Oxyrrhis marina ( o, no plastid evident27). The dinozoan branch bearing ‘replaced plastids‘ is a symbolic branch representing many such branches that obtained tertiary plastids independently. Heterotrophic dinoflagellates have characters as for Oxyrrhis marina. The tree is a consensus of data presented in this paper, and other published relationships10,12,13,28-30

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Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Acknowledgements This work was supported by: ARC grant A10009205 to DAC and OHG; ABRS grant 204-53 to DAC; grants to MO from the Czech Science Foundation grant no. 206/06/1439, Academy of Sciences of the Czech Republic project no. z60220518, and Czech Ministry of Education project no. MSMT30-801; University of Iowa startup funds and NSF grant EF-04-31117 to JML; University of Tasmania Institutional Research Grant No. B0013784 to CB. We thank Andrew McMinn for PAM data, Alastair Simpson for analytical suggestions, Bob Andersen for culture backup, Andrew Polaszek and Mark 10

Garland for taxonomic opinions, and anonymous reviewers for constructive suggestions that greatly improved the manuscript. Author Contributions RBM isolated the strain while in the DAC lab, then while in JML lab designed the AToL SSU primers and the psbA primers, cloned and sequenced multiple copies of the SSU rRNA gene, a copy of the plastid SSU rRNA gene and initial sections of the psbA and LSU rRNA genes, then assigned precedented methods for culture fixation, and wrote and finalized the draft of the paper; MO led and performed phylogenetic analyses of the sequence data, cloned and sequenced a copy of the plastid SSU rDNA gene using different primers than RBM, and co-wrote the draft of the paper; MO and MV performed the TEM and SEM data collection; JJ and TC cloned and sequenced near full length LSU rDNA and psbA genes and undertook extensive phylogenetic analysis; TC performed mito-red staining; SWW and NWD performed pigment analysis and interpreted pigment data; RBM, KH, CJSB, and JS interpreted TEM data and assigned taxonomy; KH and RBM performed light microscopy; RBM, MO, TC, KH, DHG and CJSB maintained cultures. DHG cloned and sequenced the LSU rRNA gene, utilizing different PCR primers than TC and JJ; RBM, MO, DHG, KH, JS, OHG, JML, CJB and DAC designed research, interpreted evolutionary, ecological and microbiological data, and performed extensive editing and revision. Author

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www.nature.com/reprints. Museum and genbank materials are detailed in figure captions. The authors declare no competing financial interests. Correspondence should be addressed to DAC. ([email protected]).

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Alexandrium catenella Alexandrium tamarense Alexandrium affine Alexandrium minutum Lingulodinium polyedrum DinoProrocentrum donghaiense Prorocentrum micans flagellates Pfiesteria piscicida Akashiwo sanguinea Prorocentrum micans Perkinsus chesapeki Perkinsus andrewsi Perkinsids Perkinsus olseni

Hammondia hammondi Toxoplasma gondii Neospora caninum Besnoitia besnoiti Isospora felis Frenkelia microti Sarcocystis neurona Sarcocystis muris Eimeria tenella

1

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bp np n LSU rDNA au topology 1 0.989 0.991 0.990

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Guillardia theta Porphyra purpurea Epifagus virginiana Nicotiana tabacum Chlorella ellipsoides Chlorella vulgaris Cyanothece sp. Gloeocapsa sp. Rhopalodia gibba Synechocystis sp. 0.1

0.988

0.012 0.97/64 0.91/79

Heterocapsa rotundata Heterocapsa pygmaea Scrippsiella trochoidea Alexandrium pseudogonyaulax 1.00/79 Gyrodinium dorsum Prorocentrum emarginatum 0.92/Peridinium balticum 0.95/Kryptoperidinium foliaceum Dinoflagellates Dinophysis norvegica Prorocentrum minimum Cryptoperidiniopsis brodyi 0.90/53 Gyrodinium impudicum Amphidinium semilunatum Amoebophyra sp. Eukaryotic clone OLI11005 0.99/75 Hematodinium sp. Noctiluca scintillans 0.64/98 Perkinsus atlanticus Perkinsus atlanticus Perkinsids Perkinsus sp. Perkinsus marinus Caryospora bigenetica Eimeria alabamensis 1.00/100 Toxoplasma gondii 3 Theileria buffei 1.00/Babesia gibsoni Monocystis agilis 0.99/54 Ophriocystis elektroscirrha 0.98/Selenidium terebalae Cryptosporidium serpentis 0.93/54 marine parasite from Tridacna crocea marine clone from Ammonia beccarii Environmental sequence AF372772 2 Colpodella edax 1.00/100 Environmental sequence AF372785 Colpodellids 0.99/64 Environmental sequence AF372786 0.89/56 Colpodella sp. ATCC 50594 1 Voromonas pontica 0.87/91

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0.99/58

Chromera velia

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Euglena gracillis Euglenozoans Astasia longa Dinoflagellates Karlodinium veneficum with HaptophyteKarlodinium veneficum derived plastid Isochrysis sp. Ochrosphaera sp. Haptophytes Chrysochromulina sp. Karenia sp. Dinoflagellates Karenia mikimotoi with HaptophyteKarenia mikimotoi derived plastid Karenia brevis Haptophyte Pavlova gyrans Odontella sinensis Bacillariophyte (diatom) Dinophysis acuminata Dinoflagellates with Dinophysis norvegica Cryptophyte-derived plastid Dinophysis tripos

0.68/82

Chromera velia

Urocentrum turbo Furgasonia blochmani Oxytricha nova Ciliates Didinium nasutum Protocruzia sp. 1.00/99 Blepharisma americanum Thraustochytrium multirudimentale Mallomonas striata Stramenopiles Costaria costata 0.99/83

Neospora caninum Apicoplasts Toxoplasma gondii Hyaloklossia lieberkuehni Coccidians Sarcocystis muris Eimeria tenella Eimeria sp. Haemosporidians Eimeria meleagrimitis Plasmodium falciparum Plasmodium vivax 0.99/54 Babesia bovis Babesia bigemina Piroplasmids Hepatozoon catesbianae Chromera velia Coccidian

0.56/65

0.58/77 0.92/-

Tetrahymena thermophila Tetrahymena pyriformis Ciliates Paramecium tetraurelia Nannochloropsis salina Dictyocha speculum Stramenopiles Hyphochytrium catenoides Phytophthora megasperma Guillardia theta Cryptophyte

0.65/-

b

Coccidia (Apicomplexans)

e

Cryptophyte Red alga

Plants and green algae Cyanobacteria

0.99/84 0.80/56

f 0.96/50

1

Heterocapsa triquetra Heterocapsa pygmaea Alexandrium tamarense Lingulodinium polyedrum Scrippsiella trochoidea Symbiodinium from Tridacna Thoracosphaera heimii Amphidinium carterae Prorocentrum micans

Chromera velia

0.60/-

Pylaiella littoralis Peridinin Ectocarpus siliculosus 0.61/- 2 Padina crassa dinoflagellates Dictyota pardalis Heterosigma akashiwo Stramenopiles 0.97/65 Heterosigma carterae 3 Bumilleriopsis filiformis 0.93/56 Vaucheria litorea Odontella sinensis 0.89/97 0.98/62 Isochrysis sp. Emiliana huxleyi 0.57/53 0.83/Pleurochrysis carterae Phaeocystis antarctica Haptophytes Prymnesium parvum 0.69/Pavlova lutheri Pavlova gyrans 0.90/Dinoflagellates with haptophyteKarenia brevis Karenia mikimotoi derived plastid 0.72/Palmaria palmata 0.61/Stylonema alsidii 0.69/1.00/Rhodosorus marinus Rhodophytes (red algae) 0.84/Compsopogon caeruleus 0.91/60 Porphyridium aerugineum Bangia fuscopurpurea 0.77 Rhodomonas abbreviata Cryptophytes Dinoflagellates with 0.97 Pyrenomonas helgolandii Dinophysis norvegica cryptophyte-derived Chroomonas plastid Cryptophytes Chilomonas paramecium Dixoniella grisea Rhodophytes Rhodella violacea 0.87/62

0.1

0.1

d

bp

pp

sh

wkh

wsh

topology 1

0.955 0.634

0.631

0.709 0.743

0.856

0.743

0.901

topology 2

0.150 0.351

0.358

0.291 0.257 0.610

0.257

0.491

topology 3

0.006 0.011

0.011

2e-6

0.044

0.039

0.046

n SSU rDNA

au

np

kh

0.039

g

psbA

au

topology 1

0.736

topology 2

0.334

topology 3

np

bp

pp

0.699 0.698 0.972

kh

sh

wkh

wsh

0.705

0.843 0.705

0.808

0.285 0.289 0.027 0.295 0.041 0.016 0.013 0.001 0.074

0.368 0.295

0.416

0.144 0.074

0.144

hemosporidians and piroplasms Chromera velia

plastid

X

no evidence for a plastid

X

loss of photosynthesis

R

replacement of plastid

coccidians Cryptosporidium Gregarina

colpodellids

origination of UGA-Trp readthrough

chlorophyll types a, a + c in photosynthesis UGG

UGG Trp plastid

UGG UGA

UGG+UGA Trp plastid

loss of chl c

X

other dinozoans

UGG UGA

X

X

R

peridinin dinoflagellates

a UGG

a+c

ancestor of apicomplexans and dinozoans