letters to nature 200 ng ml-1 ethidium bromide; RNA was electrophoresed on 1.2% agarose- formaldehyde gels24. Probes were labelled by a random primer system (Life Technologies) and [32P]dCTP (Amersham). Membranes were hybridized at Chlorophyll b and phycobilins 55 8C in Church buffer (0.25 M Na2HPO4, 1 mM EDTA, 7% SDS), washed with 0.1 ´ SSC/0.1% SDS at 55 8C (DNA) or 65 8C (RNA), and exposed to Kodak X- in the common ancestor Omat ®lm. DNA sequencing and analysis. Sequencing was done using a dye terminator of cyanobacteria cycle sequencing kit (Applied BioSystems); traces from the automatic sequencer were edited by Staden's Trev program (http://www.mrc-lmb.cam. and chloroplasts ac.uk/pubseq). PCR reactions (Sigma reagents) were carried out for 35 cycles: Akiko Tomitani*², Kiyotaka Okada³, Hideaki Miyashita§, 94 8C for 30 s, 55 8C for 30 s, followed by 2 min at 72 8C. PCR products puri®ed Hans C. P. Matthijsk, Terufumi Ohno¶ & Ayumi Tanaka² from 1% low-melting-agarose gels were used in sequencing. A database was set up in GAP4 of Staden and contigs were generated. We retrieved protein * Department of Geology and Mineralogy, Graduate School of Science, Kyoto University, Kyoto 6068502, Japan sequence alignments7 from 134.169.70.80/ftp/pub/incoming/; and Guillardia ² Institute of Low Temperature Science, Hokkaido University, Sapporo 0600819, sequences8 from Genbank (AF041468). Alignments were improved manually in Japan GDE (ver. 2.2)25. PHYLIP (ver. 3.5) (http://evolution.genetics.washington.edu/ ³ Department of Botany, Graduate School of Science, Kyoto University, phylip/html) was used to construct a maximum parsimony tree with global Kyoto 6068502, Japan rearrangement, and neighbour-joining trees using the PAM matrix. The input § Marine Biotechnology Institute, Kamaishi Laboratories, Kamaishi, order of taxa was jumbled. Iwate 0260001, Japan k Department of Microbiology, University of Amsterdam, Received 25 January; accepted 29 April 1999. Nieuwe Achtergracht 127, NL 1018 WS, Amsterdam, The Netherlands 1. Dodge, J. D. A survey of chloroplast ultrastructure in Dinophyceae. Phycologia 14, 253±263 (1975). ¶ The Kyoto University Museum, Kyoto University, Kyoto 6068502, Japan 2. Jeffrey, S. W. et al. Chloroplast pigment patterns in dino¯agellates. J. Phycol. 111, 374±384 (1975). ......................................................................................................................... 3. Boczar, B. A., Liston, J. & Cattolico, R. A. Characterization of satellite DNA from three marine dino¯agellates: Glenodinium sp. and two members of the toxic genus, Protogonyaulax. Plant Physiol. Photosynthetic organisms have a variety of accessory pigments, 97, 613±618 (1991). on which their classi®cation has been based. Despite this varia- 4. Gibbs, S. P. The chloroplasts of some groups of algae may have evolved from endosymbiotic eukaryotic algae. Ann. NY Acad. Sci. 361, 193±207 (1981). tion, it is generally accepted that all chloroplasts are derived 5. Cavalier-Smith, T. in Biodiversity and Evolution (eds Arai, R., Kato, M. & Doi, Y.) 75±114 (The from a single cyanobacterial ancestor1±3. How the pigment diver- National Science Museum Foundation, Tokyo, 1995). 6. Palmer, J. D. & Delwiche, C. F. in Molecular Systematics of Plants II (eds Soltis, D. E., Soltis, P. S. & sity has arisen is the key to revealing their evolutionary history. Doyle, J. J.) 375±409 (Kluwer, Norwall, MA, 1998). Prochlorophytes are prokaryotes which perform oxygenic 7. Martin, W. et al. Gene transfer to the nucleus and the evolution of chloroplasts. Nature 393, 162±165 photosynthesis using chlorophyll b, like land plants and green (1998). 8. Douglas, S. E. & Penny, S. L. The plastid genome from the cryptomonad alga, Guillardia theta: algae (Chlorophyta), and were proposed to be the ancestors of complete sequence and conserved synteny groups con®rm its common ancestry with red algae. J. Mol. chlorophyte chloroplasts4,5. However, three known prochloro- Evol. 48, 236±244 (1999). 9. Morse, D., Salois, P., Markovic, P. & Hastings, J. W. A nuclear encoded form II RuBisCo in phytes (Prochloron didemni, Prochlorothrix hollandica and dino¯agellates. Science 268, 1622±1624 (1995). Prochlorococcus marinus) have been shown to be not the speci®c 10. Rowan, R., Whitney, S. W., Fowler, A. & Yellowless, D. Rubisco in marine symbiotic dino¯agellates: ancestors of chloroplasts, but only diverged members of the form II enzymes in eukaryotic oxygenic phototrophs, encoded by a nuclear multi-gene family. Plant Cell 8, 539±553 (1996). cyanobacteria, which contain phycobilins but lack chlorophyll 11. Schlunegger, B. & Stutz, E. The Euglena gracilis chloroplast genome: structural features of a DNA b6,7. Consequently it has been proposed that the ability to synthe- region possibly carrying the single origin of DNA replication. Curr. Genet. 8, 629±634 (1984). 12. Wu, M., Lou, J. K., Chang, D. Y., Chang, C. H. & Nie, Z. Q. Structure and function of a chloroplast size chlorophyll b developed independently several times in DNA replication origin of Chlamydomonas reinhardtii. Proc. Natl Acad. Sci. USA 83, 6761±6765 prochlorophytes and in the ancestor of chlorophytes. Here we (1986). have isolated the chlorophyll b synthesis genes (chlorophyll a 13. Wakasugi, T. et al. Complete nucleotide sequence of the chloroplast genome from the green alga 8 Chlorella vulgaris: the existence of genes possibly involved in chloroplast division. Proc. Natl Acad. Sci. oxygenase) from two prochlorophytes and from major groups of USA 94, 5967±5972 (1997). chlorophytes. Phylogenetic analyses show that these genes share a 14. Boore, J. L. & Brown, W. M. Complete DNA sequence of the mitochondrial genome of the black chiton, Katharina tunicata. Genetics 138, 423±443 (1994). common evolutionary origin. This indicates that the progenitors 15. Sugiura, M., Hirose, T. & Sugita, M. Evolution and mechanism of translation in chloroplasts. Annu. of oxygenic photosynthetic bacteria, including the ancestor of Rev. Genet. 32, 437±459 (1998). chloroplasts, had both chlorophyll b and phycobilins. 16. Saunders, G. W., Hill, D. R. A., Sexton, J. P. & Andersen, R. A. Small-subunit ribosomal RNA sequences from selected dino¯agellates: testing classical evolutionary hypotheses with molecular The chlorophyll a oxygenase (CAO) gene is involved in chloro- systematic methods. Plant Syst. Evol. (suppl.) 11, 237±259 (1997). phyll b biosynthesis, and was isolated by insertional mutagenesis of 17. Jacobs, J. D. et al. Characterisation of two circular plasmids from the marine diatom Cylindrotheca 8 fusiformis: plasmids hybridise to chloroplast and nuclear DNA. Mol. Gen. Genet. 233, 302±310 (1992). the green alga Chlamydomonas reinhardtii . This gene encodes a 18. La Claire II, J. W., Loudenslager, C. M. & Zuccarello, G. C. Characterization of novel extrachromo- protein of 463 amino acids, including a putative transit peptide. The somal DNA from giant celled marine green algae. Curr. Genet. 34, 204±211 (1998). deduced amino-acid sequence contains binding domains for a [2Fe- 19. Backert, S., Nielsen, B. L. & BoÈrner, T. The mystery of the rings: structure and replication of mitochondrial genomes from higher plants. Trends Plant Sci. 2, 477±483 (1987). 2S] Rieske centre and for a mononuclear non-haem iron, indicating 20. Wilson, R. J. M. et al. Complete map of the plastid-like DNA of the malaria parasite Plasmodium that CAO encodes an oxygenase. When a CAO complementary falciparum. J. Mol. Biol. 261, 155±172 (1996). 21. Gajadhar, A. A. et al. Ribosomal RNA sequences of Sarcocystis muris, Theileria annulata and DNA was isolated from Arabidopsis thaliana and expressed in Crypthecodinium cohnii reveal evolutionary relationships among apicomplexans, dino¯agellates Escherichia coli, enzymatic study with the expressed protein and ciliates. Mol. Biochem. Parasitol. 45, 147±154 (1991). showed that conversion of chlorophyll a to chlorophyll b was carried 22. Cavalier-Smith, T. Kingdom protozoa and its 18 phyla. Microbiol. Rev. 57, 953±994 (1993). 9 23. Waller, R. et al. Nuclear-encoded proteins target to the plastid in Toxoplasma gondii and Plasmodium out by the CAO product alone . All oxygenic photosynthetic falciparum. Proc. Natl Acad. Sci. USA 98, 12352±12357 (1998). organisms contain chlorophyll a and can synthesize chlorophyll b 24. Sambrook, J., Fritsch, E. F. & Maniatis, T. Molecular Cloning, A Laboratory Manual 2nd edn (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). only by obtaining CAO. Analyses of chlorophyll b less mutants of 25. Smith, S. W. The genetic data environment and expandable GUI for multiple sequence analysis. Comp. Chlamydomonas indicate that CAO is a single-copy gene8.Comparison Appl. Biosci. 10, 671±675 (1994). of the deduced amino-acid sequences of the Chlamydomonas and Arabidopsis CAOs showed that a region of about 300 amino-acid Acknowledgements. We thank M. Beaton and K. Ishida for valuable discussion and advice; E. Filek for help with plasmid sequencing; X. Wu and E. Chao for advice on PCR; R. G. Hiller for communicating residues, which includes two motifs for a [2Fe-2S] Rieske centre and unpublished data; and J. Saldarriaga for H. rotundata and G. grindleyi total DNA. This work was for a mononuclear iron, is highly conserved. On the other hand, supported by NSERC research grants to B.R.G. and T.C.-S. T.C.-S. thanks the Canadian Institute for Advanced Research for fellowship support. both the amino- and carboxy-terminal regions have low similarity. We isolated CAO genes or cDNAs from P. hollandica,