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Phylogenetic history of plastid-targeted proteins in the peridinin-containing dinoflagellate Heterocapsa triquetra

Ross F. Waller, Nicola J. Patron and Patrick J. Keeling

Canadian Institute for Advanced Research, Department of Botany, University of British Columbia, 3529-6270 University Boulevard, Vancouver, BC, V6T 1Z4, Canada

Correspondence
Patrick J. Keeling
pkeeling{at}interchange.ubc.ca

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
The evolutionary history and relationship between plastids of dinoflagellate algae and apicomplexan parasites have been controversial both because the organelles are unusual and because their genomes contain few comparable genes. However, most plastid proteins are encoded in the host nucleus and targeted to the organelle, and several of these genes have proved to have interesting and informative evolutionary histories. We have used expressed sequence tag (EST) sequencing to generate gene sequence data from the nuclear genome of the dinoflagellate Heterocapsa triquetra and inferred phylogenies for the complete set of identified plastid-targeted proteins. Overall, dinoflagellate plastid proteins are most consistently related to homologues from the red algal plastid lineage (not green) and, in many of the most robust cases, they branch with other chromalveolate algae. In resolved phylogenies where apicomplexan data are available, dinoflagellates and apicomplexans are related. We also identified two cases of apparent lateral, or horizontal, gene transfer, one from the green plastid lineage and one from a bacterial lineage unrelated to plastids or cyanobacteria.

The GenBank/EMBL/DDBJ accession numbers for the completed cDNA sequences reported in this study are AY826826–AY826947 and AY884246–AY884255. The dbEST accession numbers for the EST sequences reported in this study are DT379484–DT386290.

Protein maximum-likelihood trees for a further 23 plastid-targeted proteins are available as supplementary material in IJSEM Online.

Present address: Botany School, University of Melbourne, Parkville, VIC 3010, Australia.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Plastids originated by the endosymbiotic uptake of a cyanobacterium and the subsequent conversion of this endosymbiont into the highly reduced and specialized double-membrane-bound primary plastid found today in land plants and some algae. Most algal groups, however, acquired their plastids by an additional step called secondary endosymbiosis. Here, an alga containing a primary plastid is itself taken up and converted into an organelle within its new eukaryotic host (Archibald & Keeling, 2002). These secondary plastids are bounded by either three or four membranes and are found in chlorarachniophytes, euglenids, cryptomonads, heterokonts, haptophytes, dinoflagellates and apicomplexans. In all of these groups, the plastid retains only a small genome: most proteins are nuclear-encoded and are targeted post-translationally to the plastid using specific N-terminal peptides that are characteristic for either primary or secondary plastids (McFadden, 2001).

Dinoflagellates are closely related to apicomplexans and, together with ciliates and a handful of other protists, make up the alveolates (Fast et al., 2002; Gajadhar et al., 1991). Since many members of both dinoflagellates and apicomplexans contain secondary plastids, the most parsimonious explanation is that they share a common origin, but the evolutionary history of both plastids has proved contentious, in part because they are divergent, making them difficult to compare.

While some dinoflagellates have undergone plastid replacements through further endosymbiotic events (Delwiche, 1999; Keeling, 2004), the plastid found in the majority of photosynthetic dinoflagellates is a secondary plastid containing the distinctive pigment peridinin. This plastid is further distinguished in many ways, not least in that it has three membranes rather than four, which appears to have a significant affect on how proteins are targeted to the organelle (Nassoury et al., 2003; Patron et al., 2005). The genomes of dinoflagellate plastids are also the most reduced known. All but a few of their genes have moved to the nucleus (Bachvaroff et al., 2004; Hackett et al., 2004a), and the remaining genome (16 genes have been found so far, nearly all related to photosynthesis) has been broken up into mini-circles each generally encoding a single gene (Zhang et al., 1999).

Apicomplexans, on the other hand, are obligate parasites, so their plastid (the apicoplast) is non-photosynthetic and generally reduced. Its relict genome contains few genes, typically highly divergent at the sequence level, and mostly encoding housekeeping functions (Wilson et al., 1996). The evolutionary origin of this plastid has been debated since its discovery, with some data interpreted as showing a green algal origin (Cai et al., 2003; Funes et al., 2002; Köhler et al., 1997) and other data interpreted as showing a red algal origin (Blanchard & Hicks, 1999; Fast et al., 2001; McFadden & Waller, 1997; Patron et al., 2004; Waller et al., 2003). Since nearly all of the genes remaining in the dinoflagellate plastid are related to photosynthesis, there are few plastid-encoded genes that can be compared directly between apicomplexans and dinoflagellates. Those that have been (primarily encoding small- and large-subunit rRNA) are highly divergent in both groups, making phylogenies difficult to interpret (Zhang et al., 2000).

The disputes over the origin of dinoflagellate and apicomplexan plastids widened with the suggestion that both plastids originated in the ancestor of all chromalveolates. The chromalveolates are a hypothetical grouping of alveolates and chromists (cryptomonads, haptophytes and heterokonts): all eukaryotes hypothesized to have red algal-derived plastids (Cavalier-Smith, 1998). Plastid-encoded gene trees have given variable results, but multigene analyses weakly unite chromist plastids (Yoon et al., 2002, 2004). Nuclear gene trees provide strong support for the alveolates and their relationship to heterokonts (Baldauf et al., 2000; Harper et al., 2005). Taken together, plastid and cytosolic data are therefore consistent with the chromalveolate hypothesis, but neither support the whole group: the strongest support for this comes from two nucleus-encoded genes for plastid-targeted proteins: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fructose-1,6-bisphosphate aldolase (FBA) (Fast et al., 2001; Harper & Keeling, 2003; Patron et al., 2004). In both cases, the chromalveolate plastid-targeted protein appears to have evolved in a unique way relative to other plastids, and these deviations suggest that plastids of chromalveolates share a common origin.

Phylogenies of GAPDH and FBA demonstrate the usefulness of nucleus-encoded plastid-targeted proteins for studying plastid evolution, but they are poorly sampled because the nuclear genome is practically less accessible than that of the plastid. In dinoflagellates, this problem is aggravated by their unusually large genome size, so expressed sequence tag (EST) surveys have been used to generate genomic data from several species of dinoflagellate (Bachvaroff et al., 2004; Hackett et al., 2004a; Patron et al., 2005). Phylogenetic analyses for some of these proteins have been carried out, some showing a substantial conflict in gene trees indicating either a red or green algal origin, or high levels of lateral gene transfer (Hackett et al., 2004a). Here, we have phylogenetically analysed all the plastid-targeted proteins identified from an EST survey of the dinoflagellate Heterocapsa triquetra (Patron et al., 2005). We have inferred phylogenies for 52 proteins, eight of which have apicomplexan homologues. Overall, the dinoflagellate plastid proteins tend to branch with red algae and chromists (with red algal secondary plastids) with stronger consistency than previously observed. Of the phylogenies containing apicomplexan homologues that are resolved with reasonable support, the apicomplexans group within the red algal plastid clade and a specific relationship with the dinoflagellate homologues was evident in some of these. Many dinoflagellate plastid-targeted proteins are relatively divergent and contain unique oddities [such as a tandem fusion of translation elongation factor Ts (EF-Ts)], and a few well-resolved phylogenies appear to support lateral gene transfer, including genes derived from prokaryotes.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
EST sequencing and protein identification.
H. triquetra CCMP 449, cultivated in Guillard's f2-Si medium at 16 °C with a 12 h : 12 h light/dark cycle, was harvested in batches and total RNA was used to construct a cDNA library as described by Patron et al. (2005). ESTs were 5'-sequenced and gene identification was performed at the PEPdb (http://amoebidia.bcm.umontreal.ca/pepdb/searches/welcome.php). Plastid-targeted proteins were identified as part of an analysis of the nature of plastid-targeting leader sequences in dinoflagellates reported in Patron et al. (2005). Briefly, EST annotation was searched for genes with known function in the plastid and the sequence database was searched using known plastid-targeted proteins from other organisms. In cases where candidate genes were determined to be full-length, they were analysed for the presence of an N-terminal leader with characteristics expected of a dinoflagellate plastid-targeting peptide (in particular the presence of a predicted signal peptide). In addition, phylogenetic analysis was carried out for all candidate genes (see below), revealing some to be related to cytosolic or mitochondrial homologues. In these cases, unless the gene encoded a leader warranting further investigation, they were no longer considered. In cases where cDNAs were truncated and the presence of a leader could not be verified, genes were considered to encode plastid-targeted proteins if they were phylogenetically related to other plastid-targeted homologues and to cyanobacterial homologues. Identification of putatively plastid-targeted proteins in apicomplexans followed previous annotation, which is based on the well-characterized leaders of annotated proteins (Ralph et al., 2004) and direct localization (e.g. Jomaa et al., 1999; Waller et al., 1998). Completed cDNA sequences have been deposited in GenBank (accession numbers AY826826–AY826947, AY884246–AY884255) and EST sequences have been deposited in dbEST (DT379484–DT386290).

Phylogenetic analysis.
Protein alignments were constructed using CLUSTAL X (Thompson et al., 1997) and edited manually. All ambiguous sites of the alignments were removed from the dataset for phylogenetic analyses. The alignment data are available on request. Protein maximum-likelihood analyses used PhyML (Guindon & Gascuel, 2003) with input trees generated by BIONJ, the JTT model of amino acids substitution, proportion of variable rates estimated from the data and nine categories of substitution rates (eight variable and one invariable). One hundred bootstrap trees were calculated with PhyML initially without gamma correction categories; if the resulting trees showed resolution, the analysis was repeated with four rate categories. For distance analyses, gamma-corrected distances were calculated by TREE-PUZZLE 5.2 (Schmidt et al., 2002) using the WAG substitution matrix with eight variable rate categories and invariable sites. Trees were inferred by weighted neighbour-joining using WEIGHBOR 1.0.1a (Bruno et al., 2000). Bootstrap resampling was performed using PUZZLEBOOT (shell script by A. Roger and M. Holder; http://www.tree-puzzle.de) with rates and frequencies estimated using TREE-PUZZLE 5.2.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Red algal origin of Heterocapsa plastid-targeted proteins
A previous analysis of plastid-targeting leaders in H. triquetra (Patron et al., 2005) identified a total of 63 distinct genes from 2022 EST clusters as likely being plastid-targeted protein-coding genes. Of these, 11 represented multiple copies of certain genes; hence there were 52 distinct plastid proteins identified in total (including several distinct lineages within the light-harvesting complex superfamily). The majority of these proteins are involved in the light or dark reactions of photosynthesis, but other activities such as transcription, translation or synthesis of fatty acids and isoprenoids were also represented, and eight proteins representing such functions had identifiable homologues in apicomplexans. These plastid proteins were each subjected to phylogenetic analyses, with the exceptions of form II ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO), which has already been analysed in detail and is known to be unique to dinoflagellates and photosynthetic proteobacteria (Whitney et al., 1995), and the light-harvesting complex proteins, which we found to form a poorly resolved protein family in dinoflagellates. Furthermore, dinoflagellate GAPDH, FBA and PsbO have been analysed previously (Fast et al., 2001; Harper & Keeling, 2003; Ishida & Green, 2002; Patron et al., 2004).

Of the 34 discrete proteins analysed (Table 1), many of the H. triquetra genes were divergent compared with other plastid homologues, and the position of H. triquetra was completely unresolved in two cases. The other 32 proteins supported H. triquetra branching with other plastid homologues, as expected. Of these, H. triquetra branched with neither the red nor green algal lineages (i.e. the position was unresolved within plastids) or, in 18 cases, plastids were polyphyletic. H. triquetra branched with the red plastid lineage (red and chromistan algae) in another 13 cases, eight with moderate to strong support, as shown in three examples in Fig. 1. In the phosphoglycerate kinase phylogeny (Fig. 1a), the H. triquetra genes for the plastid and cytosolic enzymes are both shown, and the plastid gene branches within a relatively well supported (89 %) clade of red algal and chromists genes, as well as Bigelowiella natans, which has a green algal secondary plastid and whose phosphoglycerate kinase has been proposed to be derived from a red alga by lateral gene transfer (Archibald et al., 2003). Similarly, H. triquetra phosphoribulokinase (Fig. 1b) branches specifically with chromist homologues, and most closely with the haptophyte Isochrysis (94–96 %). Lastly, H. triquetra and Alexandrium tamarense (Hackett et al., 2004a) possess a gene for photosystem II extrinsic protein (Fig. 1c), a protein apparently lost from green algae altogether and otherwise only known in cyanobacteria, red algal plastids and their chromist derivatives.

View larger version (35K): [in this window] [in a new window]   Fig. 1. Relationship of dinoflagellate plastid-targeted proteins phosphoglycerate kinase (a), queuine tRNA ribosyltransferase (b) and photosystem II extrinsic protein (c) to homologues from red algal and chromist plastids. Numbers at nodes indicate bootstrap support (ML top/left; NJ bottom/right) for major nodes over 50 % by at least one method. Major eukaryotic groups and plastids are indicated to the right.

Relationship between dinoflagellate and apicomplexan plastid-targeted proteins
Most of the H. triquetra plastid-targeted proteins are involved in photosynthesis and so are not present in apicomplexans. However, eight proteins were identified from both groups (see asterisks in Table 1), and the phylogenies of four offer some resolution (Fig. 2). The phylogenies of both dimethyladenosine synthase (Fig. 2a) and queuine tRNA ribosyltransferase (Fig. 2b) support dinoflagellates and apicomplexans as sister taxa within the red plastid clade. The latter is also of interest because it is not known from green plastids. Interestingly, however, the dinoflagellate–apicomplexan clade of queuine tRNA ribosyltransferase also includes two alphaproteobacterial sequences, the chromist plastid sequences and a Dictyostelium homologue, and there is no clear relationship between plastid genes in general and cyanobacterial homologues. Ultimately, the source of this protein in plastids is unclear: it is possible the proteobacteria and Dictyostelium each acquired this gene from plastids, but it is also possible the plastid genes are not ancestrally cyanobacterial or that there are many paralogues. In any case, the H. triquetra gene forms a strongly supported group with Toxoplasma (91–99 %), suggesting that they are mostly likely closely related, and the presence of this protein in apicomplexans is not consistent with their having a plastid of green algal ancestry. 1-Deoxy-D-xylulose-5-phosphate synthase (Fig. 2c) also places apicomplexans firmly within the red plastid clade, although the position of H. triquetra is unresolved within this red group. Apicomplexans are, nevertheless, moderately strongly allied to the chromist Thalassiosira (74–82 %) and not the green plastid lineage.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Relationship between dinoflagellate and apicomplexan plastid-targeted proteins dimethyladenosine synthase (a), queuine tRNA ribosyltransferase (b) and 1-deoxy-D-xylulose-5-phosphate synthase (c). See the legend to Fig. 1 for other details.

Evidence for lateral gene transfer and gene fusions in dinoflagellate plastid-targeted proteins
Previously it has been shown that B. natans, a chlorarachniophyte alga with a green algal secondary plastid, acquired several plastid-targeted protein genes from other algae, including red algae, or bacteria (Archibald et al., 2003). In dinoflagellates, RuBisCO has long been known to be of a bacterial type and considered to have originated by lateral gene transfer (Rowan et al., 1996; Whitney et al., 1995). One additional protein, -aminolaevulinic acid dehydratase, has also been suggested to be derived from a green alga along with other more complex cases (Hackett et al., 2004a), but the extent to which dinoflagellate plastid proteins may not originate from a single source is not well known. Other than the well-studied RuBisCO, it is similarly unclear whether any bacterial proteins have been harnessed in the plastid.

In our survey, a handful of proteins branched with the green algae or plants rather than the red plastid clade, but in all cases except one this was without support (Table 1). The one exception, the oxoglutarate/malate translocator (Fig. 3a), is interesting because, to date, the only eukaryotic sources from which it has been reported are plastids of plants and green algae. Since the preponderance of genes support the red origin of the dinoflagellate plastid, the presence of this protein in H. triquetra but not in finished genomes of red algae or diatoms (Armbrust et al., 2004; Barbier et al., 2005; Matsuzaki et al., 2004) or in any other known red or red-derived plastids suggests that this dinoflagellate protein is derived by lateral gene transfer from a green source.

View larger version (28K): [in this window] [in a new window]   Fig. 3. Phylogenies of three dinoflagellate plastid-targeted proteins with unusual evolutionary histories. (a) The oxoglutarate/malate translocator is only known in green algal plastids and now also dinoflagellates. (b) The H. triquetra acetolactate synthase is closely related to those of alphaproteobacteria and is suggested to have originated by lateral gene transfer. (c) The H. triquetra EF-Ts is a unique tandem duplication where the two halves (labelled C and N) are closely related, suggesting a recent duplication. See the legend to Fig. 1 for other details.

Lastly, one of the H. triquetra genes that branches weakly with the green algae is EF-Ts. The phylogeny of this gene (Fig. 3c) is too weak to conclude much about its origin; however, it is noteworthy because of its unique structure. The H. triquetra gene encodes a tandem duplication of EF-Ts and the phylogeny shows that the two halves of the duplication branch together with 100 % support. This close relationship between the two halves shows that the duplication took place relatively recently, which has interesting implications for the function of this protein, which recycles GDP from elongation factor-Tu during translation elongation. Several other dinoflagellate proteins are expressed as polymers, some of which are processed and some are functional fusion proteins (Hiller et al., 1995; Liu et al., 2004; Rowan et al., 1996). It is not clear whether the EF-Ts is processed or not. Despite this protein having a housekeeping role in translation, we could not identify a plastid version in apicomplexans, but we did find non-duplicated plastid homologues of EF-Ts in a diatom (Phaeodactylum) and a cryptomonad (Guillardia). The distribution of this character is relatively restricted, therefore, but seeking this protein in other apicomplexans might be of interest.

Concluding remarks
Overall, the phylogeny of dinoflagellate plastid-targeted proteins supports the origin of this organelle from the red plastid lineage. This is consistent with previous suggestions based on pigmentation (chlorophyll c is found in dinoflagellates and chromists) and some molecular data, but the analyses described here provide more consistency than previously observed with molecular phylogenetic surveys. In general, dinoflagellate proteins also tend to branch with homologues from other chromalveolates, although no single protein shows this relationship unambiguously and the best evidence for this remains the unusual evolutionary histories of GAPDH and FBA. The few plastid proteins available from both dinoflagellates and apicomplexans tend to support a common origin of these two plastids and add further evidence for the red ancestry of apicomplexan plastids. Altogether there is very little evidence to support a green origin for either dinoflagellate or apicomplexan plastids. Confirming that the dinoflagellate plastid is related to those of apicomplexans and the chromalveolates as a whole is significant for several reasons, in particular because of the many differences between dinoflagellate and apicomplexan plastids. Distinctions in membrane number and protein targeting between dinoflagellates and other chromalveolates mean that dinoflagellates must have undergone a dramatic transformation necessitating changes to the targeting system and also to the transit peptides of perhaps hundreds of proteins (Nassoury et al., 2003; Patron et al., 2005). In addition, a plastid-containing ancestor of apicomplexans and dinoflagellates is interesting because deep-branching members of these two lineages, Colpodella and Perkinsus, respectively (Goggin & Barker, 1993; Kuvardina et al., 2002), potentially enable us to reconstruct many characteristics of this ancestor and the subsequent evolution of both groups in detail (Leander & Keeling, 2003).

Analyses of plastid-targeted protein genes also continue to show the evolutionary complexity of dinoflagellate plastids, and indeed plastids as a whole. There are now a handful of dinoflagellate plastid proteins that do not seem to fit with the overall picture of the plastid's evolution from the red plastid lineage: this work and other studies (Hackett et al., 2003) have shown some proteins being more akin to green homologues and others being only distantly related to plastid homologues in general. This is similar to observations from another myxotrophic alga, B. natans (Archibald et al., 2003), raising interesting questions about the genetic content of such genomes in general. In addition, dinoflagellates seem to be prone to developing novelty at the molecular level (Hackett et al., 2004b), as evident from their plastid proteins. Complexes appear to form between proteins from distantly related sources and new structures are found, making dinoflagellates an interesting case study of molecular diversity in microbial eukaryotes.

	ACKNOWLEDGEMENTS

 
This work was supported by the Protist EST Program of Genome Canada/Genome Atlantic and by a grant (MOP-42517) from the Canadian Institutes for Health Research (CIHR). R. F. W. is supported by Fellowships from CIHR and the Michael Smith Foundation for Heath Research (MSFHR) and P. J. K. is a Fellow of the CIAR and a new investigator of the CIHR and MSFHR.

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