Response to Comment on “A Green Algal Apicoplast Ancestor” We suggested lateral transfer of split cox2a and cox2b genes from a chlorophyte algal ancestor to an apicomplexan ancestor (1). Waller et al. (2) oppose this interpretation based on a phylogeny implying close affilia- tion of apicomplexan cox2a and cox2b genes with ciliate cox2 homologues; a 900-bp in- sertion that separates the corresponding do- mains in ciliate mitochondrial cox2 genes; and low hydrophobicity values for ciliate mitochondrial COXII and apicomplexan COXIIA and COXIIB subunits. They suggest that mitochondrial cox2 genes were split and transferred to the nuclei of the apicomplexan and chlorophyte ancestors during indepen- dent events. We find their phylogenetic argument uncon- vincing because, as they admit, statistical sup- port for the position of the ciliate sequences is lacking. Affiliation of ciliates and apicomplex- ans previously noted in mitochondrial sequence phylogenies should also be interpreted with caution because both lineages are rapidly evolv- ing and may be artifactually grouped due to long-branch attraction (3). Furthermore, the ad- dition of ciliate cox2 sequences to our previous phylogenetic analysis (1), does not show a re- lationship between ciliates and apicomplexans (Fig. 1, A and B). Nevertheless, we are pleased that in agreement with our findings in (1), Waller et al. (2) confirm the common branch for apicomplexan and chlorophyte COXIIA and COXIIB sequences—an otherwise unex- pected affiliation. The presence of an insertion in the mito- chondrial cox2 genes of ciliates does not nec- essarily imply kinship with apicomplexan frag- mented cox2a and cox2b genes. The cox2 gene is susceptible to insertions between regions that encode the hydrophobic and hydrophilic do- mains of COXII. Insertions of variable lengths have been described in cox2 genes of unrelated organisms (4), including brown marine algae (NC_003055, NC_004024), microflagellates (NC_000946), and bacteria (NC_003888). Localization of insertions in cox2 is most likely constrained by the difficulty of inserting in the gene without disrupting the structure of the COXII protein (5). Ciliates also exhibit an in- sertion in cox1 and a fragmented mitochondrial nad1 (3). These rearrangements most likely occurred after the divergence of ciliates from apicomplexans and dinoflagellates (6), since no sequence remnants of insertions are present in cox1 in apicomplexans (7) or dinoflagellates (8). The cox1 and cox2 insertions were most likely absent in the alveolate ancestor and were acquired by ciliates independently. Therefore, the proposed vertical inheritance of cox2a and cox2b genes in apicomplexans is not directly supported by the presence of insertions in ciliate cox2. The hydrophobicity analysis presented by Waller et al. (2) provides no evidence for a common origin of ciliate COXII and apicom- plexan COXIIA and COXIIB sequences. We agree that hydrophobicity is one of the rate- limiting steps in functional gene transfer from the mitochondrion to the nucleus (5, 9 –11). It is therefore not surprising that apicomplexan COXIIA and COXIIB subunits exhibit di- minished hydrophobicity. Nevertheless, the low mean hydrophobicity values of ciliate mitochondrial COXII sequences merely indi- cate that, from the hydrophobicity point of view, the corresponding genes are ready to migrate—not that they migrated in the distant past. Ciliate mitochondrial DNA (mtDNA) contains more than the standard set of genes encoded by mtDNA in other eukaryotes (3) and therefore does not seem to show an in- creased rate of gene migration. The hydro- Fig. 1. Phylogenetic analysis of COXII. (A) Maximum likelihood (ML) tree. COXIIA and COXIIB (excluding MTS and extensions) were fused in silico as a single polypeptide and aligned with orthodox mitochondrial COXII sequences (14). (B) Phylogram showing branch lengths estimated with ML implementing the data, model, and parameters used to perform the ML search. COXII sequences for Tetrahymena pyriformis (NC_000862), T. thermophila (NC_003029), and Paramecium aurelia (NC_001324) were obtained from GenBank. (C) Multiple protein sequence alignment of the N-terminal regions of COXIIB sequences. Shown are the N-terminal extensions of nuclear cox2b genes from chlorophytes and apicomplexans (blue), the conserved residues in these domains (yellow), the conserved residues found only in chlorophytes and ampicomplexans (black back- ground), the corresponding region of the 300-residue insertion in ciliate mitochondrial COXII (gray), and the conserved residues between ciliates and apicomplexans (bold). Droso (Drosophila mela- nogaster), Homo (Homo sapiens), Arabi (Arabidopsis thaliana), Mesos (Mesostigam viride), Theil (Theileria parva), Plasm (Plasmodium yoelli), Toxop (Toxoplasma gondii), Chlam (Chalmydomonas reinhardtii), Polyt (Polytomella sp.), Tther (Tetrahymena termophila), Tpyri (Tetrahymena pyriformis), and Param (Paramecium aurelia). TECHNICAL COMMENT www.sciencemag.org SCIENCE VOL 301 4 JULY 2003 49b o n A p ril 5 , 2 0 2 1 h ttp ://scie n ce .scie n ce m a g .o rg / D o w n lo a d e d fro m http://science.sciencemag.org/ phobicity of contemporary ciliate COXII is unlikely to bear on either the location of its gene or apicomplexan COXIIA and COXIIB phylogeny. In addition, the hydrophobicity analysis does not argue against the proposal that the original apicomplexan mitochondrial cox2 gene was eliminated after functional acquisition of chlorophyte cox2a and cox2b sequences (1). COXIIB has a unique N-terminal exten- sion that most likely arose since the splitting of the original mitochondrial cox2 gene of the chlorophyte ancestor (1). Protein sequencing has confirmed the presence of this extension in mature chlorophyte COXIIB (5). All chlo- rophyte and apicomplexan sequences share N-terminal extensions of COXIIB containing the conserved PxxxPxxY motif not present in the canonical COXII. These extensions seem homologous and suggest a common origin for chlamydomonad and apicomplexan COXIIA and COXIIB sequences. The proposal of Waller et al. (2) would imply that this domain conservation is due to convergent evolution, which we consider unlikely. The correspond- ing borders of the ciliate COXII insertions and the apicomplexan COXIIB extensions reveal no similarities (Fig. 1C). We originally described rare characteris- tics that are shared solely between apicom- plexans and certain chlorophyte algae (1), namely, the presence of nucleus-encoded split cox2a and cox2b genes and a conserved domain present in a region of COXIIB that is not derived from orthodox COXII. Functional fragmentation of a mitochondrial gene followed by functional integration in the nucleus is an extremely rare event, unlikely to happen several times. The current evidence suggests that this phenomenon was confined to the ancestor of chlorophyte algae. Our analyses support a close relationship between apicomplexan and chlorophyte cox2a and cox2b sequenc- es specific to the mitochondrion, whereas analysis of apicoplast genome organization has suggested a red algal origin of the organelle (12). Clearly, there is still con- flicting evidence for green versus red algal ancestry in the apicomplexans (13). What- ever the outcome of this debate, the pres- ence of nucleus-encoded, fragmented cox2a and cox2b genes of green origin in apicom- plexans must be considered whenever the evolutionary story of these parasites is re- constructed. Soledad Funes Instituto de Fisiologı́a Celular Universidad Nacional Autónoma de México (UNAM) 04510 D.F., Mexico Edgar Davidson Department of Biochemistry and Molecular Pharmacology Thomas Jefferson University Philadelphia, PA 19107, USA Adrián Reyes-Prieto Instituto de Fisiologı́a Celular, UNAM Susana Magallón Instituto de Biologı́a, UNAM Pascal Herion Instituto de Investigaciones Biomédicas, UNAM Michael P. King Department of Biochemistry and Molecular Pharmacology Thomas Jefferson University Diego González-Halphen Instituto de Fisiologı́a Celular, UNAM E-mail: dhalphen@ifc.unam.mx References and Notes 1. S. Funes et al., Science 298, 2155 (2002). 2. R. F. Waller, P. J. Keeling, G. G. van Dooren, G. I. McFadden, Science 301, 49 (2003); www.sciencemag. org/cgi/content/full/301/5629/49a. 3. G. Burger et al., J. Mol. Biol. 297, 365 (2000). 4. M. P. Oudot-le Secq et al., J. Mol. Evol. 53, 80 (2001). 5. X. Pérez-Martı́nez et al., J. Biol. Chem. 276, 11302 (2001). 6. N. M. Fast et al., J. Eukaryot Microbiol. 49, 30 (2002). 7. J. E. Feagin, Int. J. Parasitol. 30, 371 (2000). 8. S. Lin et al., J. Mol. Biol. 320, 727 (2002). 9. X. Pérez-Martı́nez et al., J. Biol. Chem. 275, 30144 (2000). 10. S. Funes et al., J. Biol. Chem. 277, 6051 (2002). 11. D. O. Daley et al., Proc. Natl. Acad. Sci. U.S.A. 99, 10510 (2002). 12. B. Stoebe, K. V. Kowallik, Trends Genet. 15, 344 (1999). 13. J. D. Palmer, J. Phycol. 39, 4 (2003). 14. Sequences were aligned using ClustalX (15), with subsequent manual refinements. The corresponding aligned nucleotide sequences were used in the anal- ysis. Agrobacterium was specified as outgroup. The model that best fits the data (GTR�I� �) and its parameters (base frequency: 0.3349, 0.1732, 0.1828, 0.3091; rate matrix: 1.9883, 3.2172, 2.1240, 2.9095, 4.8212, 1.0000; proportion of invariable sites: 0.0086; shape of gamma distribution: 0.8863) were identified with Modeltest (16) and implemented in heuristic searches with TBR branch swapping, to obtain a ML tree (score of –ln L � 25559.03416). Branch support was obtained from 100 ML bootstrap replicates con- ducted under the same model and parameters as the ML search. Analyses were performed using PAUP* 4.0b10 for Macintosh and UNIX (17). 15. J. D. Thompson et al., Nucleic Acids Res. 24, 4876 (1997). 16. D. Posada, K. A. Crandall, Bioinformatics 14, 817 (1998). 17. D. L. Swofford, PAUP* 4.0: Phylogenetic Analysis Us- ing Parsimony (*and Other Methods) (Sinauer Asso- ciates, Sunderland, MA, 2002). 18 March 2003; accepted 28 May 2003 T E C H N I C A L C O M M E N T 4 JULY 2003 VOL 301 SCIENCE www.sciencemag.org49b o n A p ril 5 , 2 0 2 1 h ttp ://scie n ce .scie n ce m a g .o rg / D o w n lo a d e d fro m http://science.sciencemag.org/ Response to Comment on "A Green Algal Apicoplast Ancestor" González-Halphen Soledad Funes, Edgar Davidson, Adrián Reyes-Prieto, Susana Magallón, Pascal Herion, Michael P. 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