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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Babesia bovis: A comprehensive phylogenetic analysis of plastid-encoded genes supports 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, USA b Animal Diseases Research Unit, United States Department of Agriculture–Agricultural Research Service, Washington State University, Pullman, WA 99164-7030, USA c School of Biological Sciences and Center for Integrated Biotechnology, Washington State University, Pullman, WA 99164-4236, USA

a r t i c l e

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Article history: Received 30 April 2009 Received in revised form 23 July 2009 Accepted 24 July 2009 Available online 29 July 2009 Keywords: Apicoplasts Apicomplexans Bayesian inference Euglenozoa Red and green algae

a b s t r a c t Apicomplexan parasites commonly contain a unique, non-photosynthetic plastid-like organelle termed the apicoplast. Previous analyses of other plastid-containing organisms suggest that apicoplasts were derived from a red algal ancestor. In this report, we present an extensive phylogenetic study of apicoplast origins using multiple previously reported apicoplast sequences as well as several sequences recently reported. Phylogenetic analysis of amino acid sequences was used to determine the evolutionary origin of the organelle. A total of nine plastid genes from 37 species were incorporated in our study. The data strongly support a green algal origin for apicoplasts and Euglenozoan plastids. Further, the nearest green algae lineage to the Apicomplexans is the parasite Helicosporidium, suggesting that apicoplasts may have originated by lateral transfer from green algal parasite lineages. The results also substantiate earlier findings that plastids found in Heterokonts such as Odontella and Thalassiosira were derived from a separate secondary endosymbiotic event likely originating from a red algal lineage. Ó 2009 Elsevier Inc. All rights reserved.

1. Introduction 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 efforts (Abrahamsen et al., 2004; Brayton et al., 2007; Dunn et al., 1998; Gardner et al., 2002, 2005; Toso and Omoto, 2007a,b; Xu et al., 2004). Notably, genome sequencing has failed to detect the presence of an apicoplast genome for Cryptosporidium spp. (Abrahamsen et al., 2004; Xu et al., 2004), and ultrastructural studies indicate that the more distantly related Gregarines (Toso and Omoto, 2007a,b) and Archigregarines (Simdyanov and Kuvardina, 2007) do not appear to contain an apicoplast. While the specific role of the apicoplast in the Apicomplexan life cycle is for the most part unclear, in Plasmodium falciparum, the causative agent of malaria, the apicoplast has been demonstrated to be involved in de novo fatty acid 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 bacteria. Acquisition of the multi-walled apicoplast must have involved at least two endosymbiotic events (Keeling, 2004), and phylogenetic evidence indicates that a bacterium, probably a cyanobacterium, was the primary endosymbiont (Cavalier-Smith, * Corresponding author. Fax: +1 509 335 8529. E-mail address: [email protected] (A.O.T. Lau). 0014-4894/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2009.07.007

1992; Delwiche et al., 1995; Yoon et al., 2002). However, the identity of the secondary endosymbiont remains controversial and the phylogenetic origin of the apicoplast has been contested (Blanchard and 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 photosynthetic plastid genome have been lost or have migrated to the nucleus, resulting in much reduced genome sizes (Gray, 1992, 1993; Medlin et al., 1995). Therefore, the apicoplast genome typically encodes less than 1% of the total number of chromosomal genes. Plastid and nuclear-encoded genes such as tufA, rpo (B and C), cox2a and cox2b suggest that apicoplasts originated from green algae (Cai et al., 2003; Funes et al., 2002; Kohler et al., 1997; Williamson et al., 1994) while studies using gene order, other apicoplast(Blanchard and Hicks, 1999) or nuclear-encoded genes whose protein products are translocated to the plastid (Fast et al., 2001; Waller and McFadden, 2005) suggest that this organelle originates from red algae. Many of these studies were limited in scope due to a paucity of genetic information from a diverse selection of Apicomplexan organisms. Additional genome sequences that have recently 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) and have considerably increased the sampling density of plastidencoded genes. We have utilized comprehensive, multi-gene

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Bayesian inference analyses to determine that the apicoplast genomes originated from a green algal lineage. In addition, our results substantiate earlier findings that plastids found in Heterokonts such as Odontella and Thalassiosira were derived from a separate secondary endosymbiotic event likely originating from a red algal lineage. Odontella and Thalassiosira are types of marine diatoms with worldwide distribution. Our findings differ from the current chromalveolate hypothesis, which states that chromists and alveolates retain their plastids from a red algal ancestry.

2. Materials and methods Complete plastid sequences from a diversity of Cyanobacteria and Eukaryotes were collected from GenBank with their individual accession numbers provided in Table 1. All completely sequenced plastids from Eukaryotes were included with the exception of the land plants, where a subset of complete plastids were included. Gene overlap between these samples and Apicomplexan apicoplasts were found to include the genes clpC, LSU rRNA, SSU rRNA,

Table 1 Taxa sampled and their corresponding plastid GenBank accession number. Cyanobacteria Gloeobacter violaceus Nostoc anabaena PCC7120 Prochlorococcus marinus MIT9313 Synechococcus sp. WH8102 Thermosynechococcus elongatus BP-1

BA000045 BA000019 BX548175 BX548020 BA000039

Apicomplexans Babesia bovis T2BO Eimeria tenella Plasmodium falciparum 3D7 Theileria parva Toxoplasma gondii

AAXT00000000 AY217738 X95275/X95276 AAGK01000009 U87145

Euglenozoans Astasia longa Euglena gracilis Z

AJ294725 Z11874

Haptophyceae Emiliana huxleyi

AY741371

Cercozoa Bigelowiella natans

DQ851108

Viridiplantae Anthoceros formosae Arabidopsis thaliana Chaetosphaeridium globosum Chlamydomonas reinhardtii Chlorella vulgaris C-27 Helicosporidium sp. ex Simulium jonesii Leptosira terrestris Mesostigma viride Nephroselmis olivacea Oltmannsiellopsis viridis Oryza nivara Pseudenoclonium akinetum Scenedesmus obliqus Stigeoclonium helvetiucum

AB086179 AP00423 AF494278 BK000554 AB001684 DQ398104 EF506945 AF166114 AF137379 DQ291132 AP006728 AY835431 DQ396875 DQ630521

Heterokonts Odontella sinensis Thalassiosira pseudonana

Z67753 EF067921

Rhodophytes Cyanidioschyzon merolae Cyanidium caldarium RK1 Gracilaria tenuistipitata var. liui Porphyra purpurea

AB002583 AF022186 AY673996 U38804

Cryptophytes Guillardia theta Rhodomonas salina

AF041468 EF508371

Glaucocystophytes Cyanophora paradoxa cyanelle

U30821

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rpl14, rpl16, rpoB, rpoC1, rpoC2, rps3, rps11, rps12, and tufA. The large and small subunit rRNA were excluded for several reasons. First, the widely discussed issues of alignment of rRNA genes at this phylogenetic depth (Dacks et al., 2002; Gribaldo and Philippe, 2002) make these genes poor candidates for the current phylogenetic analysis. Second, many plastid genomes have more than one copy of the LSU and SSU while some, including Babesia and Theileria, have only one of each, complicating assessment of their homology. After initial alignments of the clpC genes, the data indicated that there were issues of orthology of this gene complicated by several apparent transfers of the orthologs to the nuclear compartment. Therefore, the clpC gene was also excluded from the analyses. Finally, some genes are missing for a few taxa, including Anthoceros: tufA; Chlamydomonas: rpoC1; Oryza: tufA; and Thalassiosira: rpl14, rpl16, rps3, rps11, and rps12. In all, this represents eight missing gene copies from 252 total possible copies for this matrix (3%). Amino acid sequence alignments were performed using a twostep process. First, amino acid sequences were compiled and aligned using Clustal X 1.83 (Huelsenbeck and Bollback, 2001; Thompson et al., 1997) with the Gonnet 250 cost matrix applied to pairwise alignments and the Gonnet series applied to the multiple alignments. Multiple amino acid alignment models were compared and these different alignment options had little effect on preliminary analyses (data not shown). Alignment results suggested that some regions of the genes were much less conserved than others, with significantly greater amounts of inferred indel events in these regions. Due to the uncertainty of the alignments in these gene regions, Gblocks (Castresana, 2000; Talavera and Castresana, 2007) was used to select those regions of the aligned sequences that are confidently aligned for analysis. Gblocks eliminates poorly aligned positions and divergent regions of an alignment of DNA or protein sequences and selects sequence segments that lack large segments of contiguous non-conserved positions, lack of gap positions and high conservation of flanking positions. Maximum likelihood (ML) analyses of the complete and truncated nucleotide matrices were performed using PAUP* 4.0b10 and heuristic searches were employed with the starting tree obtained via neighbor-joining (NJ) and using the tree-bisectionreconnection (TBR) branch swapping algorithm (Swofford et al., 2001). Clade support was estimated using 100 heuristic bootstrap replicates using a reduced data set (four Viridiplantae, one Haptophyceae and one Cercozoa were omitted as compared to the final set of taxa included in the Bayesian analysis). Results from the ML were congruent with the final Bayesian results, thus none of the ML data were shown to avoid redundancy in the report. Bayesian inference analysis was performed on the Gblocks individual and combined-gene matrices using MrBayes v.3.0 (Huelsenbeck and Bollback, 2001). Seven and a half million generations were run with four chains (Markov Chain Monte Carlo), the heating parameter set at 0.05, and a tree was saved every 1000 generations. Priors for all analyses included the mixed amino acid model implementing a covarion model, as applied in MrBayes. The covarion model allows for rates to change across the topology (Galtier, 2001; Huelsenbeck et al., 2002; Tuffley and Steel, 1998). In order to test for the occurrence of stationarity, convergence, and mixing within 7.5 million generations, multiple analyses were started from different random locations in tree space. The posterior probability distributions from these separate replicates were compared for convergence to the same posterior probabilities across branches. Majority rule consensus trees of those sampled in Bayesian inference analyses yielded probabilities that the clades are monophyletic (Lewis, 2001). The trees from the MrBayes analysis were loaded into PAUP* 4.0b10 (Swofford et al., 2001), discarding the trees generated within the first 2,000,000 generations (those

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sampled during the ‘‘burnin” of the chain (Huelsenbeck and Ronquist, 2001), to only include trees after stationarity was established. Posterior probability values (pp) are presented on a sample tree from the post-stationarity distribution of Bayesian trees in order to demonstrate branch lengths.

3. Results and discussion Babesia bovis, an Apicomplexan hemoparasite, is one of the most prevalent tick-borne pathogens of cattle worldwide. Results from the B. bovis genome sequencing project revealed the presence of a circular 33 kbp plastid-like genome (Brayton et al., 2007). Although the function of apicoplasts is not well established, an investigation into its origin will no doubt provide insight into Apicomplexans’ evolution and gene loss in parasites (Keeling, 2004). B. bovis apicoplast genome together with the recent availability of Theileria apicoplast and Helicosporidium plastid sequences allowed us to conduct a comprehensive phylogenetic analysis of apicoplast/plastid genomes using nine genes common to all apicoplast/plastid genomes, including rpl14, rpl16, rpoB, rpoC1, rpoC2, rps3, rps11, rps12, and tufA. Analyses were conducted on deduced amino acid sequence alignments, and these were analyzed individually as well as together in a combined analysis (Figs. 1 and 2). All of the genes are located on nonrecombining plastids, and therefore, share the same history. These genes were analyzed for five Cyanobacteria, one Glaucocystophyte, two Cryptophyte, four red algae, two Heterokonts, 14 green plants (five Streptophytes and nine Chlorophytes), two Euglenozoans, one Haptophyceae, one Cercozoa, and five Apicomplexans (Table 1). Although the Apicomplexan, Sarcocystis muris, has been reported to contain an apicoplast, sequences for this organelle are not currently available. Cryptosporidium hominis and C. parvum appear to have lost their apicoplast and the associated genome, as genes of plastid origin were detected in the nuclear genome but no contiguous apicoplast sequence was found in the complete genome (Abrahamsen et al., 2004; Wilson et al., 1996; Xu et al., 2004; Zhu et al., 2000). Gregarina niphandrodes and Selenidium orientale also appear to have lost their apicoplast genomes (Toso and Omoto, 2007a,b). It has been suggested that sequences from the apicoplast genome itself should not be used to determine its phylogenetic position due to a high AT content that drives long branch attraction towards otherwise distantly related lineages (Keeling, 2004). However, where amino acid (or DNA) changes have been properly modeled and analyzed under a likelihood framework, these influences should be minimized. In addition, we used a covarion model that specifically corrects for rate variation across the tree, further minimizing any potential for a long branch attraction effect from AT content variation. The suggestion that high AT content in apicoplast genomes improperly forces this lineage towards the green algae (Morton, 1999) is not supported by the similar AT content in both red and green algae. Last but not least, additional analyses excluding the Euglenozoa plastid genes, which have a highly biased AT content, were also conducted in our study (data not shown) and resulted in the same topologies as in Fig. 1. Consequently, we consider the combined data set results in these analyses as the best estimate of relationships of eukaryotic plastids without high AT content skewing the overall outcome. In this study, the analysis of 2826 amino acid (AA) combinedgene with Gblocks matrix resulted in a robust phylogenetic hypothesis for apicoplast origins and so this combined data set is used to represent our consensus hypothesis (Fig. 1). Individualgene analysis with Gblocks matrices varied in length from 115 to 854 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 individual matrices (Fig. 2A–I) resulted in generally congruent topolo-

gies, although often with less resolution and lower posterior probability support for branches. The statistical support of these relationships is very high as evidenced by the number of branches with posterior probability values greater than 95%. Independent MrBayes analyses that were performed converged on the same posterior probability distribution of trees, suggesting that convergence and mixing were occurring in these analyses. Shorter analyses (5 million generations) with 8 chains also converged similarly and fully resolved the consensus topology. Therefore, our results suggest that (i) the plastids in the Heterokonts Odontella and Thalassiosira originated separately from the Apicomplexans and are likely derived from a red algal lineage (Fig. 1) and (ii) that the apicoplast and the Euglenozoan plastids were similarly derived from green plant lineages. Overall patterns found here propose that the roots of the Eukaryotic plastids are in the vicinity of the Glaucocystophyceae and red algae. Strong branch support is found for the placement of the Apicomplexans with the Euglenozoa and Viridiplantae (Chlorophyta and Streptophyta). Further, the Guillardia plastid is found to originate from the red algae, as previously suggested (Hagopian et al., 2004) and finally, the Chlorophytes and Streptophytes, as traditionally delimited, do not resolve into monophyletic groups (Fig. 1). Previous analysis of the phylogenetic position of the Plasmodium apicoplast suggested that some data supported a red algal origin, but that a combined analysis of all genes supported a green algal origin (Blanchard and Hicks, 1999). The phylogenetic hypotheses presented in that study did not include estimates of branch support and, therefore, how strongly one of these topologies was supported over the other was unclear. Analysis of the individual genes in our study generally did not provide strong support for many of the internal branches of the trees, regardless of the topology found. These results differ from a recent review of plastid origins (Keeling, 2004) in which it is suggested that both the Heterokonts and Apicomplexans are derived through secondary endosymbiosis from red algae (the chromalveolate hypothesis), whereas the Euglenozoan plastid is derived from a green algal source. Within the Apicomplexans, Babesia and Theileria are sister lineages, as would be expected given their similar plastid genome organization, and these taxa together are sister to Plasmodium (Fig. 1). Eimeria and Toxoplasma form a sister clade to this group of three. Furthermore, the parasitic green algae Helicosporidium is strongly placed as sister to the Apicomplexans in the combined analysis (Fig. 1), and this relationship shows support in the rps3, rps11, rps12, rpoB, and rpoC1 individual-gene analyses (Fig. 2). Results from several studies have been used as a basis for the chromalveolate hypothesis. For example, red algal origins of the apicoplast have been suggested using phylogenetic analysis of nuclear-encoded, plastid-targeted GAPDH genes (Fast et al., 2001). Three aspects of that study should be noted. First, the authors use a complex model of derivation of these nuclear-encoded genes to explain why the plastid-targeted copies are more closely related to eukaryotic cytosolic copies than to plastid copies from plants, red or green algae. It is not clear why these results favor red algal origins, and while it appears that both the Apicomplexans and Dinoflagellates have undergone gene replacement, this does not directly address the origins of the plastids to which their nuclear-encoded gene products are targeted. Second, the branching structure of the phylogeny presented had very low statistical support for most branches, limiting confidence that the presented tree reflects true branching relationships. Third, since nuclear genomes evolve at different rates than those of the plastid (Lynch, 1997; Martin, 1999), the accuracy of nuclear copies of formerly plastid-encoded genes to represent plastid origins is questionable. Gene order and plastid structure have also been used to address the phylogenetic position of the apicoplast (Blanchard

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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 represent branch 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.

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

Theileria is further complicated by the change from a doublestranded coding structure seen in most plastids to a singlestranded coding arrangement for all genes in the apicoplast genomes of these two taxa.

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Fig. 2. Bayesian inference analyses of individual-gene Gblocks amino acid alignments. Posterior probabilities are denoted at each node when 50% or greater. (A) rpl14 consensus 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.

Other studies have supported a green algal origin for apicoplasts. Cai et al.’s analysis of rpoB, rpoC1, and rpoC2 genes using maximum likelihood and Bayesian inference methods similarly provide strong statistical support for this using a smaller sample of Apicomplexans and Heterokonts (Cai et al., 2003). A recent paper on the phylogenetic position of the red algae Gracilaria tenuistipitata var. liui analyzed the relationships among photosynthetic organisms using 41 plastid protein-coding 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 supported and the inferred relationships of photosynthetic taxa were very similar to our results (Hagopian et al., 2004) (Fig. 1). If Apicomplexan sequences were improperly placed in the current study, we would expect larger perturbations of the overall tree structure. Several lines of evidence also showed the inconsistency of a single origin of the plastid in all Chromista and Alveolata (Bodyl, 2004).

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Fig. 2 (continued)

The invertebrate pathogen, Helicosporidium, is a green alga which has retained a non-photosynthetic plastid (Tartar and Boucias, 2004; Tartar et al., 2002). This taxon groups very strongly with Euglenozoans and Apicomplexans (Fig. 1), and resolves as the sister lineage of the Apicomplexans. The close phylogenetic position

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

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pling within the Trebouxiophyceae green algae may lead to a better understanding of which green algal lineage contributed the plastid to Apicomplexans, and will help to better define when the apicoplast became non-photosynthetic. Nonetheless, this analysis is the most comprehensive to date, including the most taxa and plastid genes, and uses rigorous Bayesian inference analyses of all data sets. These analyses provide strong multi-gene statistical support for the green algal hypothesis. Understanding the origins of plastids across eukaryotic lineages is critical to understanding overall patterns of diversification, mechanisms of innovation in these lineages, and may play an important role in understanding ecological 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 Medicine, Washington State University. References

Fig. 2 (continued)

Chromera velia, was recently reported to be closely related to the Apicomplexans although it retains a photosynthetic plastid (Moore et al., 2008). This conclusion was based on analyzing the nuclear large subunit rDNA sequences and the psbA gene. Since this study utilized nuclear-encoded genes to infer relationship, we caution that nuclear genes cannot be routinely used to predict the rate of evolution of an organelle, as evolution rates of nuclear and apicoplast genes can be very different as their genome evolution are governed by different events (Lynch, 1997; Martin, 1999). Thus, nuclear genes that were once encoded by the apicoplast could evolve at different rates than those that remained in the apicoplast. The C. velia study also reported the UGA-Trp usage in psbA and claimed 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 only holds true for Neospora caninum and T. gondii. UGG-Trp is still preferentially used in E. tenella (a coccidian), and other Apicomplexans such as, T. parva and B. bovis (data not shown). Last but not least, the study reported the detection of an isofucoxanthin isomer in C. velia and this implies that its plastid is of red algal origin. This last finding is intriguing and adds to the already contested debate of the apicoplast origin. Based on our analysis, we conclude that there is strong support for a green algal origin of Apicomplexan and Euglenozoan plastids, in contrast to the plastid origins of Heterokonts, which were likely derived from secondary endosymbiosis of red algae (Fig. 1). It is, however, plausible that our conclusion of green algal origin of apicoplast could be due to the possibility that the ancestral host which gave rise to the (red) apicoplast contained some green plastid genes (Cai et al., 2003). It should also be noted that sampling density of taxa can have a large influence on phylogenetic inferences, and while we have substantially increased the sampling density of Apicomplexans and Heterokonts in our study, understanding the precise patterns of secondary endosymbiotic events in all plastid-containing organisms will require much more detailed sampling of the green and red algal lineages. Further sam-

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