Babesia bovis: A comprehensive phylogenetic analysis of plastid-encoded genes supports green algal origin of apicoplasts

Experimental Parasitology 123 (2009) 236–243

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Experimental Parasitology

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Babesia bovis: A comprehensive phylogenetic analysis of plastid-encoded genessupports green algal origin of apicoplasts

Audrey O.T. Lau a,*, Terry F. McElwain a, Kelly A. Brayton a, Donald P. Knowles a,b, Eric H. Roalson c

a Program in Genomics, Department of Veterinary Microbiology & Pathology, School for Global Animal Health, College of Veterinary Medicine,Washington State University, Pullman, WA 99164-7040, USAb Animal Diseases Research Unit, United States Department of Agriculture–Agricultural Research Service, Washington State University, Pullman, WA 99164-7030, USAc School of Biological Sciences and Center for Integrated Biotechnology, Washington State University, Pullman, WA 99164-4236, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 30 April 2009Received in revised form 23 July 2009Accepted 24 July 2009Available online 29 July 2009

Keywords:ApicoplastsApicomplexansBayesian inferenceEuglenozoaRed and green algae

0014-4894/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.exppara.2009.07.007

* Corresponding author. Fax: +1 509 335 8529.E-mail address: [email protected] (A.O.T. Lau)

Apicomplexan parasites commonly contain a unique, non-photosynthetic plastid-like organelle termedthe apicoplast. Previous analyses of other plastid-containing organisms suggest that apicoplasts werederived from a red algal ancestor. In this report, we present an extensive phylogenetic study of apicoplastorigins using multiple previously reported apicoplast sequences as well as several sequences recentlyreported. Phylogenetic analysis of amino acid sequences was used to determine the evolutionary originof the organelle. A total of nine plastid genes from 37 species were incorporated in our study. The datastrongly support a green algal origin for apicoplasts and Euglenozoan plastids. Further, the nearest greenalgae lineage to the Apicomplexans is the parasite Helicosporidium, suggesting that apicoplasts may haveoriginated by lateral transfer from green algal parasite lineages. The results also substantiate earlier find-ings that plastids found in Heterokonts such as Odontella and Thalassiosira were derived from a separatesecondary endosymbiotic event likely originating from a red algal lineage.

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1. Introduction 1992; Delwiche et al., 1995; Yoon et al., 2002). However, the iden-

Since the first genome report in Plasmodium (Gardner et al.,2002), apicoplast genomes have been detected in Theileria, Eimeria,Toxoplasma, and Babesia through complete genome sequencing ef-forts (Abrahamsen et al., 2004; Brayton et al., 2007; Dunn et al.,1998; Gardner et al., 2002, 2005; Toso and Omoto, 2007a,b; Xuet al., 2004). Notably, genome sequencing has failed to detect thepresence of an apicoplast genome for Cryptosporidium spp. (Abra-hamsen et al., 2004; Xu et al., 2004), and ultrastructural studiesindicate that the more distantly related Gregarines (Toso and Omot-o, 2007a,b) and Archigregarines (Simdyanov and Kuvardina, 2007)do not appear to contain an apicoplast. While the specific role ofthe apicoplast in the Apicomplexan life cycle is for the most part un-clear, in Plasmodium falciparum, the causative agent of malaria, theapicoplast has been demonstrated to be involved in de novo fattyacid synthesis (Waller et al., 2003). This biosynthetic pathway,which is considered a novel chemotherapeutic target (Gornicki,2003), is identical to those utilized in plant chloroplasts and bacte-ria. Acquisition of the multi-walled apicoplast must have involvedat least two endosymbiotic events (Keeling, 2004), andphylogenetic evidence indicates that a bacterium, probably acyanobacterium, was the primary endosymbiont (Cavalier-Smith,

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tity of the secondary endosymbiont remains controversial and thephylogenetic origin of the apicoplast has been contested (Blanchardand Hicks, 1999; Cai et al., 2003; Fast et al., 2001; Funes et al., 2002;Kohler et al., 1997; Waller et al., 2003; Waller and McFadden, 2005;Williamson et al., 1994; Zhang et al., 1999).

Most of the genes encoded by the ancestral photosyntheticplastid genome have been lost or have migrated to the nucleus,resulting in much reduced genome sizes (Gray, 1992, 1993; Medlinet al., 1995). Therefore, the apicoplast genome typically encodesless than 1% of the total number of chromosomal genes. Plastidand nuclear-encoded genes such as tufA, rpo (B and C), cox2a andcox2b suggest that apicoplasts originated from green algae (Caiet al., 2003; Funes et al., 2002; Kohler et al., 1997; Williamsonet al., 1994) while studies using gene order, other apicoplast-(Blanchard and Hicks, 1999) or nuclear-encoded genes whoseprotein products are translocated to the plastid (Fast et al., 2001;Waller and McFadden, 2005) suggest that this organelle originatesfrom red algae. Many of these studies were limited in scope due toa paucity of genetic information from a diverse selection of Api-complexan organisms. Additional genome sequences that haverecently become available include Theileria parva (Gardner et al.,2005), T. annulata (Pain et al., 2005), Babesia bovis (Brayton et al.,2007), and Thalassiosira pseudonana (Armbrust et al., 2004) andhave considerably increased the sampling density of plastid-encoded genes. We have utilized comprehensive, multi-gene

A.O.T. Lau et al. / Experimental Parasitology 123 (2009) 236–243 237

Bayesian inference analyses to determine that the apicoplast gen-omes originated from a green algal lineage. In addition, our resultssubstantiate earlier findings that plastids found in Heterokontssuch as Odontella and Thalassiosira were derived from a separatesecondary endosymbiotic event likely originating from a red algallineage. Odontella and Thalassiosira are types of marine diatomswith worldwide distribution. Our findings differ from the currentchromalveolate hypothesis, which states that chromists and alveo-lates retain their plastids from a red algal ancestry.

2. Materials and methods

Complete plastid sequences from a diversity of Cyanobacteriaand Eukaryotes were collected from GenBank with their individualaccession numbers provided in Table 1. All completely sequencedplastids from Eukaryotes were included with the exception of theland plants, where a subset of complete plastids were included.Gene overlap between these samples and Apicomplexan apicop-lasts were found to include the genes clpC, LSU rRNA, SSU rRNA,

Table 1Taxa sampled and their corresponding plastid GenBank accession number.

CyanobacteriaGloeobacter violaceus BA000045Nostoc anabaena PCC7120 BA000019Prochlorococcus marinus MIT9313 BX548175Synechococcus sp. WH8102 BX548020Thermosynechococcus elongatus BP-1 BA000039

ApicomplexansBabesia bovis T2BO AAXT00000000Eimeria tenella AY217738Plasmodium falciparum 3D7 X95275/X95276Theileria parva AAGK01000009Toxoplasma gondii U87145

EuglenozoansAstasia longa AJ294725Euglena gracilis Z Z11874

HaptophyceaeEmiliana huxleyi AY741371

CercozoaBigelowiella natans DQ851108

ViridiplantaeAnthoceros formosae AB086179Arabidopsis thaliana AP00423Chaetosphaeridium globosum AF494278Chlamydomonas reinhardtii BK000554Chlorella vulgaris C-27 AB001684Helicosporidium sp. ex Simulium jonesii DQ398104Leptosira terrestris EF506945Mesostigma viride AF166114Nephroselmis olivacea AF137379Oltmannsiellopsis viridis DQ291132Oryza nivara AP006728Pseudenoclonium akinetum AY835431Scenedesmus obliqus DQ396875Stigeoclonium helvetiucum DQ630521

HeterokontsOdontella sinensis Z67753Thalassiosira pseudonana EF067921

RhodophytesCyanidioschyzon merolae AB002583Cyanidium caldarium RK1 AF022186Gracilaria tenuistipitata var. liui AY673996Porphyra purpurea U38804

CryptophytesGuillardia theta AF041468Rhodomonas salina EF508371

GlaucocystophytesCyanophora paradoxa cyanelle U30821

rpl14, rpl16, rpoB, rpoC1, rpoC2, rps3, rps11, rps12, and tufA. Thelarge and small subunit rRNA were excluded for several reasons.First, the widely discussed issues of alignment of rRNA genes atthis phylogenetic depth (Dacks et al., 2002; Gribaldo and Philippe,2002) make these genes poor candidates for the current phyloge-netic analysis. Second, many plastid genomes have more thanone copy of the LSU and SSU while some, including Babesia andTheileria, have only one of each, complicating assessment of theirhomology. After initial alignments of the clpC genes, the data indi-cated that there were issues of orthology of this gene complicatedby several apparent transfers of the orthologs to the nuclear com-partment. Therefore, the clpC gene was also excluded from theanalyses. Finally, some genes are missing for a few taxa, includingAnthoceros: tufA; Chlamydomonas: rpoC1; Oryza: tufA; and Thalass-iosira: rpl14, rpl16, rps3, rps11, and rps12. In all, this representseight missing gene copies from 252 total possible copies for thismatrix (�3%).

Amino acid sequence alignments were performed using a two-step process. First, amino acid sequences were compiled andaligned using Clustal X 1.83 (Huelsenbeck and Bollback, 2001;Thompson et al., 1997) with the Gonnet 250 cost matrix appliedto pairwise alignments and the Gonnet series applied to the multi-ple alignments. Multiple amino acid alignment models were com-pared and these different alignment options had little effect onpreliminary analyses (data not shown). Alignment results sug-gested that some regions of the genes were much less conservedthan others, with significantly greater amounts of inferred indelevents in these regions. Due to the uncertainty of the alignmentsin these gene regions, Gblocks (Castresana, 2000; Talavera and Cas-tresana, 2007) was used to select those regions of the aligned se-quences that are confidently aligned for analysis. Gblockseliminates poorly aligned positions and divergent regions of analignment of DNA or protein sequences and selects sequence seg-ments that lack large segments of contiguous non-conserved posi-tions, lack of gap positions and high conservation of flankingpositions.

Maximum likelihood (ML) analyses of the complete and trun-cated nucleotide matrices were performed using PAUP* 4.0b10and heuristic searches were employed with the starting tree ob-tained via neighbor-joining (NJ) and using the tree-bisection-reconnection (TBR) branch swapping algorithm (Swofford et al.,2001). Clade support was estimated using 100 heuristic bootstrapreplicates using a reduced data set (four Viridiplantae, one Hapto-phyceae and one Cercozoa were omitted as compared to the finalset of taxa included in the Bayesian analysis). Results from theML were congruent with the final Bayesian results, thus none ofthe ML data were shown to avoid redundancy in the report. Bayes-ian inference analysis was performed on the Gblocks individualand combined-gene matrices using MrBayes v.3.0 (Huelsenbeckand Bollback, 2001). Seven and a half million generations wererun with four chains (Markov Chain Monte Carlo), the heatingparameter set at 0.05, and a tree was saved every 1000 generations.Priors for all analyses included the mixed amino acid model imple-menting a covarion model, as applied in MrBayes. The covarionmodel allows for rates to change across the topology (Galtier,2001; Huelsenbeck et al., 2002; Tuffley and Steel, 1998). In orderto test for the occurrence of stationarity, convergence, and mixingwithin 7.5 million generations, multiple analyses were startedfrom different random locations in tree space. The posterior prob-ability distributions from these separate replicates were comparedfor convergence to the same posterior probabilities acrossbranches. Majority rule consensus trees of those sampled in Bayes-ian inference analyses yielded probabilities that the clades aremonophyletic (Lewis, 2001). The trees from the MrBayes analysiswere loaded into PAUP* 4.0b10 (Swofford et al., 2001), discardingthe trees generated within the first 2,000,000 generations (those

238 A.O.T. Lau et al. / Experimental Parasitology 123 (2009) 236–243

sampled during the ‘‘burnin” of the chain (Huelsenbeck and Ron-quist, 2001), to only include trees after stationarity was estab-lished. Posterior probability values (pp) are presented on asample tree from the post-stationarity distribution of Bayesiantrees in order to demonstrate branch lengths.

3. Results and discussion

Babesia bovis, an Apicomplexan hemoparasite, is one of the mostprevalent tick-borne pathogens of cattle worldwide. Results fromthe B. bovis genome sequencing project revealed the presence of acircular 33 kbp plastid-like genome (Brayton et al., 2007). Althoughthe function of apicoplasts is not well established, an investigationinto its origin will no doubt provide insight into Apicomplexans’ evo-lution and gene loss in parasites (Keeling, 2004). B. bovis apicoplastgenome together with the recent availability of Theileria apicoplastand Helicosporidium plastid sequences allowed us to conduct a com-prehensive phylogenetic analysis of apicoplast/plastid genomesusing nine genes common to all apicoplast/plastid genomes, includ-ing rpl14, rpl16, rpoB, rpoC1, rpoC2, rps3, rps11, rps12, and tufA. Anal-yses were conducted on deduced amino acid sequence alignments,and these were analyzed individually as well as together in a com-bined analysis (Figs. 1 and 2). All of the genes are located on non-recombining plastids, and therefore, share the same history. Thesegenes were analyzed for five Cyanobacteria, one Glaucocystophyte,two Cryptophyte, four red algae, two Heterokonts, 14 green plants(five Streptophytes and nine Chlorophytes), two Euglenozoans, oneHaptophyceae, one Cercozoa, and five Apicomplexans (Table 1).Although the Apicomplexan, Sarcocystis muris, has been reportedto contain an apicoplast, sequences for this organelle are not cur-rently available. Cryptosporidium hominis and C. parvum appear tohave lost their apicoplast and the associated genome, as genes ofplastid origin were detected in the nuclear genome but no contigu-ous apicoplast sequence was found in the complete genome (Abra-hamsen et al., 2004; Wilson et al., 1996; Xu et al., 2004; Zhu et al.,2000). Gregarina niphandrodes and Selenidium orientale also appearto have lost their apicoplast genomes (Toso and Omoto, 2007a,b).

It has been suggested that sequences from the apicoplast gen-ome itself should not be used to determine its phylogenetic posi-tion due to a high AT content that drives long branch attractiontowards otherwise distantly related lineages (Keeling, 2004). How-ever, where amino acid (or DNA) changes have been properly mod-eled and analyzed under a likelihood framework, these influencesshould be minimized. In addition, we used a covarion model thatspecifically corrects for rate variation across the tree, further min-imizing any potential for a long branch attraction effect from ATcontent variation. The suggestion that high AT content in apicop-last genomes improperly forces this lineage towards the green al-gae (Morton, 1999) is not supported by the similar AT content inboth red and green algae. Last but not least, additional analysesexcluding the Euglenozoa plastid genes, which have a highlybiased AT content, were also conducted in our study (data notshown) and resulted in the same topologies as in Fig. 1. Conse-quently, we consider the combined data set results in these analy-ses as the best estimate of relationships of eukaryotic plastidswithout high AT content skewing the overall outcome.

In this study, the analysis of 2826 amino acid (AA) combined-gene with Gblocks matrix resulted in a robust phylogenetichypothesis for apicoplast origins and so this combined data set isused to represent our consensus hypothesis (Fig. 1). Individual-gene analysis with Gblocks matrices varied in length from 115 to854 amino acids (rpl14 – 115 AA; rpl16 – 132 AA; rpoB – 854 AA;rpoC1 – 433 AA; rpoC2 – 509 AA; rps3 – 147 AA; rps11 – 116 AA;rps12 – 121 AA; and tufA – 399 AA). Bayesian analysis of these indi-vidual matrices (Fig. 2A–I) resulted in generally congruent topolo-

gies, although often with less resolution and lower posteriorprobability support for branches. The statistical support of theserelationships is very high as evidenced by the number of brancheswith posterior probability values greater than 95%. IndependentMrBayes analyses that were performed converged on the sameposterior probability distribution of trees, suggesting that conver-gence and mixing were occurring in these analyses. Shorter analy-ses (5 million generations) with 8 chains also converged similarlyand fully resolved the consensus topology. Therefore, our resultssuggest that (i) the plastids in the Heterokonts Odontella and Tha-lassiosira originated separately from the Apicomplexans and arelikely derived from a red algal lineage (Fig. 1) and (ii) that the api-coplast and the Euglenozoan plastids were similarly derived fromgreen plant lineages.

Overall patterns found here propose that the roots of the Eukary-otic plastids are in the vicinity of the Glaucocystophyceae and red al-gae. Strong branch support is found for the placement of theApicomplexans with the Euglenozoa and Viridiplantae (Chlorophytaand Streptophyta). Further, the Guillardia plastid is found to origi-nate from the red algae, as previously suggested (Hagopian et al.,2004) and finally, the Chlorophytes and Streptophytes, as tradition-ally delimited, do not resolve into monophyletic groups (Fig. 1).

Previous analysis of the phylogenetic position of the Plasmo-dium apicoplast suggested that some data supported a red algalorigin, but that a combined analysis of all genes supported a greenalgal origin (Blanchard and Hicks, 1999). The phylogenetic hypoth-eses presented in that study did not include estimates of branchsupport and, therefore, how strongly one of these topologies wassupported over the other was unclear. Analysis of the individualgenes in our study generally did not provide strong support formany of the internal branches of the trees, regardless of thetopology found. These results differ from a recent review of plas-tid origins (Keeling, 2004) in which it is suggested that both theHeterokonts and Apicomplexans are derived through secondaryendosymbiosis from red algae (the chromalveolate hypothesis),whereas the Euglenozoan plastid is derived from a green algalsource. Within the Apicomplexans, Babesia and Theileria are sis-ter lineages, as would be expected given their similar plastidgenome organization, and these taxa together are sister to Plas-modium (Fig. 1). Eimeria and Toxoplasma form a sister clade tothis group of three. Furthermore, the parasitic green algae Heli-cosporidium is strongly placed as sister to the Apicomplexansin the combined analysis (Fig. 1), and this relationship showssupport in the rps3, rps11, rps12, rpoB, and rpoC1 individual-geneanalyses (Fig. 2).

Results from several studies have been used as a basis for thechromalveolate hypothesis. For example, red algal origins of theapicoplast have been suggested using phylogenetic analysis of nu-clear-encoded, plastid-targeted GAPDH genes (Fast et al., 2001).Three aspects of that study should be noted. First, the authorsuse a complex model of derivation of these nuclear-encoded genesto explain why the plastid-targeted copies are more closely relatedto eukaryotic cytosolic copies than to plastid copies from plants,red or green algae. It is not clear why these results favor red algalorigins, and while it appears that both the Apicomplexans andDinoflagellates have undergone gene replacement, this does not di-rectly address the origins of the plastids to which their nuclear-en-coded gene products are targeted. Second, the branching structureof the phylogeny presented had very low statistical support formost branches, limiting confidence that the presented tree reflectstrue branching relationships. Third, since nuclear genomes evolveat different rates than those of the plastid (Lynch, 1997; Martin,1999), the accuracy of nuclear copies of formerly plastid-encodedgenes to represent plastid origins is questionable.

Gene order and plastid structure have also been used to ad-dress the phylogenetic position of the apicoplast (Blanchard

Fig. 1. Bayesian inference analysis of the combined-gene Gblocks amino acid alignment. Relationships represented by one of the post-burnin topologies in order to representbranch lengths. Posterior probabilities are denoted at each node when 50% or greater. Branches marked by ‘‘//” have been reduced in scale by 50% in order to fit the page.

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and Hicks, 1999). These comparisons are difficult to interpret,particularly given the extreme levels of gene loss in most ofthe secondary endosymbionts. Using gene order as a tool forcomparison of the origin of the apicoplasts from Babesia and

Theileria is further complicated by the change from a double-stranded coding structure seen in most plastids to a single-stranded coding arrangement for all genes in the apicoplast gen-omes of these two taxa.

Fig. 2. Bayesian inference analyses of individual-gene Gblocks amino acid alignments. Posterior probabilities are denoted at each node when 50% or greater. (A) rpl14consensus tree. (B) rpl16 consensus tree. (C) rps3 consensus tree. (D) rps11 consensus tree. (E) rps12 consensus tree. (F) rpoB consensus tree. (G) rpoC1 consensus tree. (H)rpoC2 consensus tree. (I) tufA consensus tree.

240 A.O.T. Lau et al. / Experimental Parasitology 123 (2009) 236–243

Other studies have supported a green algal origin for apicop-lasts. Cai et al.’s analysis of rpoB, rpoC1, and rpoC2 genes usingmaximum likelihood and Bayesian inference methods similarlyprovide strong statistical support for this using a smaller sam-ple of Apicomplexans and Heterokonts (Cai et al., 2003). A re-cent paper on the phylogenetic position of the red algaeGracilaria tenuistipitata var. liui analyzed the relationshipsamong photosynthetic organisms using 41 plastid protein-cod-ing genes (Hagopian et al., 2004). While the Euglenozoa and

Apicomplexan plastids were not included in this analysis,branching structure in the Gracilaria study was strongly sup-ported and the inferred relationships of photosynthetic taxawere very similar to our results (Hagopian et al., 2004)(Fig. 1). If Apicomplexan sequences were improperly placed inthe current study, we would expect larger perturbations ofthe overall tree structure. Several lines of evidence also showedthe inconsistency of a single origin of the plastid in all Chromi-sta and Alveolata (Bodyl, 2004).

Fig. 2 (continued)

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The invertebrate pathogen, Helicosporidium, is a green algawhich has retained a non-photosynthetic plastid (Tartar and Bou-cias, 2004; Tartar et al., 2002). This taxon groups very strongly withEuglenozoans and Apicomplexans (Fig. 1), and resolves as the sis-ter lineage of the Apicomplexans. The close phylogenetic position

of this green alga to the Apicomplexans further strengthens theargument that the apicoplast is of green algal origin. Interestingly,the association of the apicoplast with a non-photosynthetic greenalga raises the question of when the plastid became non-photosynthetic.

Fig. 2 (continued)

242 A.O.T. Lau et al. / Experimental Parasitology 123 (2009) 236–243

Chromera velia, was recently reported to be closely related tothe Apicomplexans although it retains a photosynthetic plastid(Moore et al., 2008). This conclusion was based on analyzing thenuclear large subunit rDNA sequences and the psbA gene. Since thisstudy utilized nuclear-encoded genes to infer relationship, we cau-tion that nuclear genes cannot be routinely used to predict the rateof evolution of an organelle, as evolution rates of nuclear and api-coplast genes can be very different as their genome evolution aregoverned by different events (Lynch, 1997; Martin, 1999). Thus,nuclear genes that were once encoded by the apicoplast couldevolve at different rates than those that remained in the apicoplast.The C. velia study also reported the UGA-Trp usage in psbA andclaimed to be a feature also found in apicoplasts of coccidians(Lang-Unnasch and Aiello, 1999) and mitochondria (Ralph et al.,2004). This observation of the unusual UGA-Trp codon usage onlyholds true for Neospora caninum and T. gondii. UGG-Trp is still pref-erentially used in E. tenella (a coccidian), and other Apicomplexanssuch as, T. parva and B. bovis (data not shown). Last but not least,the study reported the detection of an isofucoxanthin isomer inC. velia and this implies that its plastid is of red algal origin. Thislast finding is intriguing and adds to the already contested debateof the apicoplast origin.

Based on our analysis, we conclude that there is strong supportfor a green algal origin of Apicomplexan and Euglenozoan plastids,in contrast to the plastid origins of Heterokonts, which were likelyderived from secondary endosymbiosis of red algae (Fig. 1). It is,however, plausible that our conclusion of green algal origin of api-coplast could be due to the possibility that the ancestral hostwhich gave rise to the (red) apicoplast contained some green plas-tid genes (Cai et al., 2003). It should also be noted that samplingdensity of taxa can have a large influence on phylogenetic infer-ences, and while we have substantially increased the samplingdensity of Apicomplexans and Heterokonts in our study, under-standing the precise patterns of secondary endosymbiotic eventsin all plastid-containing organisms will require much more de-tailed sampling of the green and red algal lineages. Further sam-

pling within the Trebouxiophyceae green algae may lead to abetter understanding of which green algal lineage contributedthe plastid to Apicomplexans, and will help to better define whenthe apicoplast became non-photosynthetic. Nonetheless, this anal-ysis is the most comprehensive to date, including the most taxaand plastid genes, and uses rigorous Bayesian inference analysesof all data sets. These analyses provide strong multi-gene statisticalsupport for the green algal hypothesis. Understanding the originsof plastids across eukaryotic lineages is critical to understandingoverall patterns of diversification, mechanisms of innovation inthese lineages, and may play an important role in understandingecological roles of (and possibly biological control of) these cryptic‘‘protists”.

Acknowledgments

This work was supported by USDA-ARS SCA58-5348-2-683,SCA5348-32000-020-01S and CRIS project 5348-32000-020-00D,and the Animal Health Research Center, College of Veterinary Med-icine, Washington State University.

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