*Canadian Institute for Advanced Research, Department of Botany, University of British Columbia, Vancouver, British Columbia, Canada;
Department of Biology, University of Pennsylvania
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Abstract |
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Introduction |
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In recent years, apicomplexa have been found to harbor a relic, nonphotosynthetic plastid homologous to the chloroplasts of plants and algae. This plastid, or apicoplast, was first identified in Plasmodium and Toxoplasma (McFadden et al. 1996
; Wilson et al. 1996
; Köhler et al. 1997
) but now appears to be widespread throughout the phylum (Denny et al. 1998
; Lang-Unnasch et al. 1998
). Ultrastructural and molecular data have confirmed that this organelle originated by secondary endosymbiosis, whereby a heterotrophic eukaryote engulfed and retained a photosynthetic eukaryote, resulting in a plastid with more than two bounding membranes (Waller et al. 1998
). Although the apicomplexan plastid has been postulated to be surrounded by three membranes (Hopkins et al. 1999
), other studies show it to be surrounded by four membranes (Köhler et al. 1997
): two inner membranes corresponding to the plastid membranes of the primary endosymbiont, a third membrane which is thought to be the plasma membrane of the secondary endosymbiont, and an outermost membrane which is homologous to the host endomembrane system. Protein trafficking to secondary plastids is complicated by these extra membranes, so proteins encoded by genes in the host nucleus that are targeted to the secondary plastid require a signal peptide to direct the product to the endomembrane system and a transit peptide to direct the protein into the plastid, unlike plastid-targeted products in plants, which require only a transit peptide (Delwiche 1999
; McFadden 1999
). Nuclear-encoded, plastid-targeted genes from both Toxoplasma and Plasmodium have been found to contain such bipartite leader sequences, and targeting studies with green fluorescent protein in Toxoplasma and Plasmodium also indicate that the bipartite leader is necessary and sufficient to target proteins to the plastid (Waller et al. 1998, 2000
).
The presence of a plastid in apicomplexa has attracted considerable attention, primarily for two reasons. First, the plastid is a remarkable target for therapeutics, since plastid metabolic pathways are prokaryotic in origin, allowing herbicides and antibiotics to specifically disrupt the parasite with minimal effect on the animal host (Jomaa et al. 1999
; McFadden and Roos 1999
; Zuther et al. 1999
). Second, the origin of this plastid has been something of an evolutionary enigma, since it is unclear why obligate intracellular parasites would acquire or retain a plastid when they are clearly not photosynthetic. Arguments based on gene order in plastid genomes and on some plastid gene phylogenies have been used to support a red algal origin for the apicomplexan plastid, but other phylogenies (based on the translation factor tufA) have alternatively been used to support the notion that the secondary symbiont was a green alga (Williamson et al. 1994
; Köhler et al. 1997
; McFadden and Waller 1997
). To complicate matters further, the apicomplexa are now well known to share a very close relationship with dinoflagellates, and dinoflagellates also contain a plastid (here we refer specifically to the peridinin-containing plastid of dinoflagellates) derived from secondary endosymbiosis in this case involving a red alga (Zhang, Green, and Cavalier-Smith 1999
). The apicomplexan-dinoflagellate relationship has raised questions as to whether the apicoplast shares a common origin with the dinoflagellate plastid; however, a solution to this problem has proved difficult, since plastid gene sequences from both groups are extremely divergent and not phylogenetically informative (Zhang, Green, and Cavalier-Smith 2000
).
We addressed the evolutionary origin of the apicomplexan plastid using a novel approach. In both groups, nuclear-encoded, plastid-targeted gene sequences are generally less divergent than plastid-encoded sequences and are therefore more amenable to phylogenetic analysis. We accordingly characterized the first comparable plastid-targeted genes from apicomplexa and dinoflagellates, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and used these genes to test the relationship between their plastids. We sequenced cytosolic copies of GAPDH from the apicomplexan Toxoplasma gondii, the ciliates Tetrahymena thermophila, Paramecium tetraurelia, Halteria grandinella, and Blepharisma intermedium, and the heterokont alga Heterosigma akashiwo. Plastid-targeted GAPDH sequences were also determined from T. gondii and H. akashiwo.
GAPDH is a central metabolic enzyme involved in both glycolysis and the Calvin cycle. Phylogenetic analyses of GAPDH sequences have proven to be notoriously complex, reflecting a complicated history of gene duplications, lateral transfers, and endosymbiotic gene replacements (e.g., Martin et al. 1993
; Henze et al. 1995
; Liaud et al. 1997
; Figge et al. 1999
). Previously characterized plastid-targeted GAPDH genes from a dinoflagellate were found to be most closely related to cryptomonad plastid-targeted genes, but together these plastid genes are clearly related to eukaryotic cytosolic genes (Fagan, Hastings, and Morse 1998
). In contrast, the plastid GAPDH genes from plants, green algae, and red algae are closely related to cyanobacteria and other eubacteria, as one would expect of a plastid-derived gene, suggesting that the dinoflagellate and cryptomonad plastid-targeted GAPDH genes originated by lateral gene transfer or endosymbiotic gene replacement (Liaud et al. 1997
; Fagan, Hastings, and Morse 1998
). Because of this unusual evolutionary history, we can test whether the apicoplast and dinoflagellate plastids are related simply by determining whether the apicomplexan plastid-targeted GAPDH is specifically related to the peculiar dinoflagellate gene or to the cyanobacteria-plastid lineage.
Phylogenetic trees constructed with our new sequences from apicomplexa and their more distant relatives ciliates and heterokonts show that the apicomplexan plastid-targeted GAPDH gene is indeed related to dinoflagellate plastid-targeted GAPDH, supporting the conclusion that their plastids arose from a single secondary endosymbiotic event involving a red alga. In addition, both genes are part of a very highly supported clade that also includes plastid-targeted GAPDH from heterokonts and cryptomonads, two other groups with red algal secondary endosymbionts, altogether suggesting that the plastids of all these groups are homologous and arose from a single engulfment of a red alga. This evidence confirms the red algal origin of the apicoplast and shows that the apicoplast is related to the dinoflagellate plastid. Moreover, it also suggests that this endosymbiosis took place relatively early in eukaryotic evolution, leading to the provocative possibility that ciliates at one time also harbored a plastid.
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Materials and Methods |
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Near-full-length GAPDH sequences were amplified from H. akashiwo, B. intermedium, P. tetraurelia (strain 51.s), and H. grandinella by PCR using the degenerate primers 5'-CCAAGGTCGGNATHAAYGGNTTYGG and 5'-CGAGTAGCCCCAYTCRTTRTCRTACCA. All amplification reactions consisted of 35 cycles of 92°C for 12 s, 45°C for 12 s, and 72°C for 1 min with a slope of 3.0 in an Idaho Rapidcycler. Products were cloned into the TOPO-TA vector pCR2.1 (Invitrogen), and multiple clones of each were sequenced on both strands with ABI BigDye terminator chemistry. Heterosigma akashiwo, B. intermedium, P. tetraurelia, and H. grandinella DNAs were generous gifts from K. Ishida, J. Berger, J. Preer, and D. Lynn, respectively. The T. thermophila GAPDH sequence was amplified from an excised UniZap cDNA library generously provided by A. Turkewitz. The 5' end of the T. thermophila GAPDH was amplified from the same cDNA preparation using the M13 Reverse primer in conjunction with an exact-match primer designed to the 5' end of the T. thermophila GAPDH.
New sequences have been deposited in GenBank under accession numbers AF319448AF319456, AF265361, and AF265362.
Phylogenetic Analysis
Amino acid alignments were created with PIMA (Smith and Smith 1992
), and the alignment was edited manually. Distances were calculated from 290 unambiguously aligned characters by PUZZLE, version 4.0.1 (Strimmer and von Haeseler 1996
), using the JTT substitution matrix with the frequency of amino acid usage calculated from the data. Distances were calculated assuming both a constant rate of substitution and modeling site-to-site variation on a gamma curve with eight rate categories, estimating invariable sites and the shape parameter from the data. Trees were constructed with BioNJ (Gascuel 1997
) and Fitch-Margoliash (Felsenstein 1993
), with the latter using global rearrangements and 10 input order jumbles. The same well-supported groups were seen in analyses with both constant and gamma-corrected rates, in both BioNJ and Fitch-Margoliash trees. All trees shown are BioNJ trees. Bootstrap trees were constructed using both BioNJ and Fitch-Margoliash (with no global rearrangements or input order jumbles) from 100 resampled data sets, with gamma-corrected distances (with the rate category parameters above) calculated using Puzzleboot (shell script by M. Holder and A. Roger).
Kishino-Hasegawa tests (Kishino and Hasegawa 1989
) to assess alternative tree topologies were performed using PUZZLE, version 4.0.1. Rate category parameters were as above, and alternatives were deemed significantly worse at a 5% or 1% level.
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Results |
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As ciliates are members of the alveolates, and therefore close relatives of apicomplexa and dinoflagellates (Van de Peer and De Wachter 1997
), GAPDH gene sequences were also amplified from the ciliates Halteria, Blepharisma, Paramecium, and Tetrahymena. The ciliate alternative genetic code (Caron and Meyer 1985
; Preer et al. 1985
) is evident by the presence of in-frame TAR codons in the GAPDH genes of Halteria, Paramecium, and Tetrahymena. Paramecium genes are also typified by short spliceosomal introns (Russell, Fraga, and Hinrichsen 1994
), and 2327-bp introns were found in the GAPDH sequences of Paramecium. The Paramecium GAPDH sequence is very similar to a short (279 bp) sequence fragment in the database, and the T. thermophila gene sequence is very similar to the partial (515 bp) Tetrahymena pyriformis sequence in GenBank (Hafid et al. 1998
) (two other T. pyriformis GAPDH fragments in the database appear to arise from bacterial contaminants).
To determine if the ciliate GAPDH genes possessed a leader sequence, the 5' end of the GAPDH gene was amplified from T. thermophila cDNA. Several different cDNA ends from the same gene were sequenced and none encoded a leader; in all cases, transcripts contained a 5060-bp 5' UTR followed by a methionine codon corresponding to the amino terminus of the mature GAPDH coding sequence. These data are congruent with previous analysis of a different Tetrahymena GAPDH cDNA that is described in the literature but is not available in GenBank (Zhao et al. 1997
).
Molecular data indicate that heterokonts are also potential relatives of alveolates (Van de Peer and De Wachter 1997
; Tengs et al. 2000
), and since heterokonts also contain secondary endosymbiotic plastids, GAPDH was amplified from the genomic DNA of H. akashiwo, resulting in two different GAPDH sequences. Comparing these sequences with those available in the database indicated that each was similar to either the plastid-targeted GAPDH or the cytosolic GAPDH of diatoms, which were reported during the course of this work (see Liaud et al. [2000]
, which also reports the presence of a mitochondrion-targeted TPI-GAPDH fusion protein in heterokonts that is not related to the GAPDH described here).
Phylogenetic Analysis
New GAPDH sequences were aligned with available GAPDH sequences in GenBank, and a global phylogenetic analysis was undertaken based on a data set of 290 unambiguously aligned characters. The resulting tree (fig. 1
) recovered the relationships typical of GAPDH phylogeny; eukaryotic cytosolic GAPDH (GapC) is clearly distinct from the main lineage of eubacterial GAPDH (GapA/B), which includes the plastid-targeted GAPDH genes of land plants, green algae, and red algae. The apicomplexan plastid-targeted GAPDH does not branch with these plastid sequences, but instead branches with eukaryotic GapC genes, specifically in the clade containing the dinoflagellate, heterokont, and cryptomonad plastid-targeted GAPDH (supported at 100% by bootstrap analysis). The inclusion of these genes in the eukaryotic GapC clade at the exclusion of the "typical" plastid-targeted GapA/B genes is very strongly supported by bootstrap analysis (at several highly supported nodes), altogether suggesting that the apicomplexan plastid-targeted GAPDH is indeed derived from the same endosymbiotic event as that of dinoflagellates. Interestingly, these plastid-targeted GAPDH genes are most closely related to the cytosolic GAPDH genes from these same organisms (with the exclusion of cryptomonads), which is consistent with the notion that the plastid-targeted GAPDH originated through an endosymbiotic gene replacement involving a duplication of the cytosolic homolog. Although it is a formal possibility that the plastid-targeted GAPDH could have arisen from the algal nuclear lineage (i.e., a transfer of the nuclear GAPDH of the secondary symbiont), this is not supported by the phylogeny (see the positions of the algal cytosolic sequences in figs. 1 and 2
).
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In contrast to the strong support for the plastid-targeted clade, there is little bootstrap support for the node uniting the alveolate and heterokont cytosolic GAPDH sequences shown in figure 2
; nor is there support for the internal relationships within this clade. However, this clade is supported by the presence of a two-amino-acid insertion which is unique to this group (fig. 2 ), with the single exception of the Heterosigma plastid homolog, which appears to be the result of a recombination event between the plastid and cytosolic paralogs in Heterosigma. There is no other evidence of recombination from examining the alignment, but this is not unexpected given the presumed ancient nature of this event. Also in contrast to the plastid-targeted clade, the cytosolic clade does not include the cryptomonad cytosolic GAPDHs, which branch with animals in figures 1 and 2
. The relationship of the cryptomonad and dinoflagellate plastid-targeted GAPDH genes has previously been attributed to lateral transfer (Fagan, Hastings, and Morse 1998
), although a relationship between cryptomonads, alveolates, and heterokonts has also been proposed (Cavalier-Smith 1999
; Liaud et al. 2000
). Kishino-Hasegawa tests were used to compare 12 alternative placements of the cryptomonad cytosolic GAPDHs, and these tests showed that their exact position among GapCs was not clear, although their placement with the cytosolic GAPDHs from alveolates and heterokonts was rejected at the 5% level. Clearly, the plastid-targeted and cytosolic homologs of cryptomonads are telling conflicting stories, but presently it is not possible to discern which of the two genes, if either, was involved in a transfer.
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Discussion |
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Previously characterized dinoflagellate plastid-targeted GAPDH genes are not specifically related to the plastid GAPDH genes of plants, green algae, or red algae, which are all of cyanobacterial origin, but are instead derived from eukaryotic cytosolic GAPDH (Fagan, Hastings, and Morse 1998
). This eukaryotic origin of the dinoflagellate plastid-targeted gene is indicative of endosymbiotic gene replacement, in this case, the plastid homolog being replaced by a nuclear, cytosolic homolog. Endosymbiotic gene replacements are not uncommon in plastid-bearing lineages (either in cases such as this or in cases in which plastid genes replace cytosolic homologs) and have affected several different genes, including plastid GAPDH genes among gymnosperms and ferns (Meyer-Gauen et al. 1994
). The eukaryotic origin of the dinoflagellate plastid GAPDH allowed us to test the relationship between the apicoplast and dinoflagellate plastids by determining whether the apicomplexan plastid-targeted GAPDH shares this distinctive position with the dinoflagellates (which would show that the plastids shared a common origin) or whether it branches with the plastid sequences of plants, green algae, and red algae (which would show that the apicomplexan and dinoflagellate plastids arose independently).
In addition to characterizing plastid and cytosolic GAPDH sequences from Toxoplasma, we also sequenced plastid and cytosolic genes from the heterokont, Heterosigma, and cytosolic GAPDHs from several ciliates. Phylogenetic trees with these new sequences showed that the apicomplexan plastid-targeted sequence did indeed branch very strongly with the clade that included the dinoflagellate plastid-targeted GAPDH sequences, along with the plastid-targeted GAPDHs of heterokonts and cryptomonads, both of which also contained red algal secondary endosymbionts. Moreover, this clade was weakly but specifically related to the cytosolic GAPDH genes of apicomplexa, dinoflagellates, ciliates, and heterokonts, lending further support to the notion that the plastid-targeted GAPDH in these organisms arose by a duplication of the cytosolic gene, at least in the ancestor of heterokonts and alveolates (how the cryptomonads fit into this picture is not certain, and lateral transfer has been evoked to explain their plastid-targeted GAPDH). Altogether, the simplest explanation for this phylogeny is depicted in figure 3A.
Both the plastid-targeted and the cytosolic clades support a close relationship between alveolate and heterokont nuclear lineages (which is also supported by several other molecular phylogenies, e.g., Van de Peer and De Wachter 1997
). The gene duplication that resulted in the cytosolic and plastid-targeted clades must have occurred prior to the divergence of alveolates and heterokonts (and perhaps cryptomonads). Since all of the plastid-targeted GAPDH genes are closely related, it follows that the plastid also originated before this divergence and that one of the duplicate GAPDH genes was targeted to the plastid. The lineages would then have diverged, and ciliates later lost their plastid. For illustrative purposes, an alternative explanation, whereby these secondary plastids arose independently, is outlined in figure 3B.
Here, the gene duplication still has to predate the divergence of the groups; however, in this scenario, cryptomonads, heterokonts, apicomplexa, and dinoflagellates all have to independently undergo a secondary symbiotic event (most or all with a red alga), and all have to independently "choose" to target the same GAPDH paralog to the secondary plastid. We reject this alternative not simply on the grounds of parsimony, but also on the grounds that the shear coincidence of independent symbioses with red algae and independent targeting of the same paralog is entirely unlikely. Similarly, the retention of both paralogs through a considerable span of evolutionary history, presumably with no function in a nonplastid-containing cell (since ciliates apparently do not retain this gene) is also extremely implausible.
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While these issues regarding the apicoplast are important, implications of plastid-targeted GAPDH phylogeny are also more far-reaching, since the origin of this secondary plastid likely far predates the divergence of apicomplexa and dinoflagellates. A single origin for the heterokont, alveolate, and perhaps cryptomonad plastid means that plastid loss must have occurred extensively among the heterokonts, and intriguingly suggests that ciliates have a photosynthetic ancestry. There is the provocative possibility that other evidence for a plastid still exists in ciliates, perhaps in the form of transferred genes or even an as-yet-unrecognized organelle. Regardless, it is clear that this single endosymbiotic event has had a tremendous effect on eukaryotic evolution.
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Conclusions |
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Acknowledgements |
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Footnotes |
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1 Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; UTR, untranslated region.
2 Keywords: apicomplexa
dinoflagellates
plastid
evolution
alveolates
3 Address for correspondence and reprints: Naomi M. Fast, Department of Botany, University of British Columbia, 3529-6270 University Boulevard, Vancouver, BC V6T 1Z4, Canada. E-mail: nfast{at}interchange.ubc.ca
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