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 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 article info abstract

Article history: Apicomplexan parasites commonly contain a unique, non-photosynthetic plastid-like organelle termed Received 30 April 2009 the apicoplast. Previous analyses of other plastid-containing organisms suggest that apicoplasts were Received in revised form 23 July 2009 derived from a red algal ancestor. In this report, we present an extensive phylogenetic study of apicoplast Accepted 24 July 2009 origins using multiple previously reported apicoplast sequences as well as several sequences recently Available online 29 July 2009 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 Keywords: strongly support a green algal origin for apicoplasts and Euglenozoan plastids. Further, the nearest green Apicoplasts algae lineage to the Apicomplexans is the parasite Helicosporidium, suggesting that apicoplasts may have Apicomplexans Bayesian inference originated by lateral transfer from green algal parasite lineages. The results also substantiate earlier ﬁnd- Euglenozoa ings that plastids found in Heterokonts such as Odontella and Thalassiosira were derived from a separate Red and green algae secondary endosymbiotic event likely originating from a red algal lineage. Ó 2009 Elsevier Inc. All rights reserved.

1. Introduction 1992; Delwiche et al., 1995; Yoon et al., 2002). However, the iden- tity of the secondary endosymbiont remains controversial and the Since the ﬁrst genome report in Plasmodium (Gardner et al., phylogenetic origin of the apicoplast has been contested (Blanchard 2002), apicoplast genomes have been detected in Theileria, Eimeria, and Hicks, 1999; Cai et al., 2003; Fast et al., 2001; Funes et al., 2002; Toxoplasma, and Babesia through complete genome sequencing ef- Kohler et al., 1997; Waller et al., 2003; Waller and McFadden, 2005; forts (Abrahamsen et al., 2004; Brayton et al., 2007; Dunn et al., Williamson et al., 1994; Zhang et al., 1999). 1998; Gardner et al., 2002, 2005; Toso and Omoto, 2007a,b; Xu Most of the genes encoded by the ancestral photosynthetic et al., 2004). Notably, genome sequencing has failed to detect the plastid genome have been lost or have migrated to the nucleus, presence of an apicoplast genome for Cryptosporidium spp. (Abra- resulting in much reduced genome sizes (Gray, 1992, 1993; Medlin hamsen et al., 2004; Xu et al., 2004), and ultrastructural studies et al., 1995). Therefore, the apicoplast genome typically encodes indicate that the more distantly related Gregarines(Toso and Omot- less than 1% of the total number of chromosomal genes. Plastid o, 2007a,b) and Archigregarines (Simdyanov and Kuvardina, 2007) and nuclear-encoded genes such as tufA, rpo (B and C), cox2a and do not appear to contain an apicoplast. While the speciﬁc role of cox2b suggest that apicoplasts originated from green algae (Cai the apicoplast in the Apicomplexan life cycle is for the most part un- et al., 2003; Funes et al., 2002; Kohler et al., 1997; Williamson clear, in Plasmodium falciparum, the causative agent of malaria, the et al., 1994) while studies using gene order, other apicoplast- apicoplast has been demonstrated to be involved in de novo fatty (Blanchard and Hicks, 1999) or nuclear-encoded genes whose acid synthesis (Waller et al., 2003). This biosynthetic pathway, protein products are translocated to the plastid (Fast et al., 2001; which is considered a novel chemotherapeutic target (Gornicki, Waller and McFadden, 2005) suggest that this organelle originates 2003), is identical to those utilized in plant chloroplasts and bacte- from red algae. Many of these studies were limited in scope due to ria. Acquisition of the multi-walled apicoplast must have involved a paucity of genetic information from a diverse selection of Api- at least two endosymbiotic events (Keeling, 2004), and complexan organisms. Additional genome sequences that have phylogenetic evidence indicates that a bacterium, probably a recently become available include Theileria parva (Gardner et al., cyanobacterium, was the primary endosymbiont (Cavalier-Smith, 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 plastid- * Corresponding author. Fax: +1 509 335 8529. E-mail address: [email protected] (A.O.T. Lau). encoded genes. We have utilized comprehensive, multi-gene

Bayesian inference analyses to determine that the apicoplast gen- rpl14, rpl16, rpoB, rpoC1, rpoC2, rps3, rps11, rps12, and tufA. The omes originated from a green algal lineage. In addition, our results large and small subunit rRNA were excluded for several reasons. substantiate earlier findings that plastids found in Heterokonts First, the widely discussed issues of alignment of rRNA genes at such as Odontella and Thalassiosira were derived from a separate this phylogenetic depth (Dacks et al., 2002; Gribaldo and Philippe, secondary endosymbiotic event likely originating from a red algal 2002) make these genes poor candidates for the current phyloge- lineage. Odontella and Thalassiosira are types of marine diatoms netic analysis. Second, many plastid genomes have more than with worldwide distribution. Our findings differ from the current one copy of the LSU and SSU while some, including Babesia and chromalveolate hypothesis, which states that chromists and alveo- Theileria, have only one of each, complicating assessment of their lates retain their plastids from a red algal ancestry. homology. After initial alignments of the clpC genes, the data indi- cated that there were issues of orthology of this gene complicated by several apparent transfers of the orthologs to the nuclear com- 2. Materials and methods partment. Therefore, the clpC gene was also excluded from the analyses. Finally, some genes are missing for a few taxa, including Complete plastid sequences from a diversity of Cyanobacteria Anthoceros: tufA; Chlamydomonas: rpoC1; Oryza: tufA; and Thalass- and Eukaryotes were collected from GenBank with their individual iosira: rpl14, rpl16, rps3, rps11, and rps12. In all, this represents accession numbers provided in Table 1. All completely sequenced eight missing gene copies from 252 total possible copies for this plastids from Eukaryotes were included with the exception of the matrix (3%). land plants, where a subset of complete plastids were included. Amino acid sequence alignments were performed using a two- Gene overlap between these samples and Apicomplexan apicop- step process. First, amino acid sequences were compiled and lasts were found to include the genes clpC, LSU rRNA, SSU rRNA, 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 multi- Table 1 Taxa sampled and their corresponding plastid GenBank accession number. ple alignments. Multiple amino acid alignment models were compared and these different alignment options had little effect on Cyanobacteria preliminary analyses (data not shown). Alignment results sug- Gloeobacter violaceus BA000045 Nostoc anabaena PCC7120 BA000019 gested that some regions of the genes were much less conserved Prochlorococcus marinus MIT9313 BX548175 than others, with significantly greater amounts of inferred indel Synechococcus sp. WH8102 BX548020 events in these regions. Due to the uncertainty of the alignments Thermosynechococcus elongatus BP-1 BA000039 in these gene regions, Gblocks (Castresana, 2000; Talavera and Cas- Apicomplexans tresana, 2007) was used to select those regions of the aligned se- Babesia bovis T2BO AAXT00000000 quences that are confidently aligned for analysis. Gblocks Eimeria tenella AY217738 Plasmodium falciparum 3D7 X95275/X95276 eliminates poorly aligned positions and divergent regions of an Theileria parva AAGK01000009 alignment of DNA or protein sequences and selects sequence seg- Toxoplasma gondii U87145 ments that lack large segments of contiguous non-conserved posi- Euglenozoans tions, lack of gap positions and high conservation of flanking Astasia longa AJ294725 positions. Euglena gracilis Z Z11874 Maximum likelihood (ML) analyses of the complete and trun- Haptophyceae cated nucleotide matrices were performed using PAUP* 4.0b10 Emiliana huxleyi AY741371 and heuristic searches were employed with the starting tree ob- Cercozoa tained via neighbor-joining (NJ) and using the tree-bisection- Bigelowiella natans DQ851108 reconnection (TBR) branch swapping algorithm (Swofford et al., Viridiplantae 2001). Clade support was estimated using 100 heuristic bootstrap Anthoceros formosae AB086179 replicates using a reduced data set (four Viridiplantae, one Hapto- Arabidopsis thaliana AP00423 phyceae and one Cercozoa were omitted as compared to the final Chaetosphaeridium globosum AF494278 Chlamydomonas reinhardtii BK000554 set of taxa included in the Bayesian analysis). Results from the Chlorella vulgaris C-27 AB001684 ML were congruent with the final Bayesian results, thus none of Helicosporidium sp. ex Simulium jonesii DQ398104 the ML data were shown to avoid redundancy in the report. Bayes- Leptosira terrestris EF506945 ian inference analysis was performed on the Gblocks individual Mesostigma viride AF166114 Nephroselmis olivacea AF137379 and combined-gene matrices using MrBayes v.3.0 (Huelsenbeck Oltmannsiellopsis viridis DQ291132 and Bollback, 2001). Seven and a half million generations were Oryza nivara AP006728 run with four chains (Markov Chain Monte Carlo), the heating Pseudenoclonium akinetum AY835431 parameter set at 0.05, and a tree was saved every 1000 generations. Scenedesmus obliqus DQ396875 Priors for all analyses included the mixed amino acid model imple- Stigeoclonium helvetiucum DQ630521 menting a covarion model, as applied in MrBayes. The covarion Heterokonts model allows for rates to change across the topology (Galtier, Odontella sinensis Z67753 Thalassiosira pseudonana EF067921 2001; Huelsenbeck et al., 2002; Tuffley and Steel, 1998). In order to test for the occurrence of stationarity, convergence, and mixing Rhodophytes Cyanidioschyzon merolae AB002583 within 7.5 million generations, multiple analyses were started Cyanidium caldarium RK1 AF022186 from different random locations in tree space. The posterior prob- Gracilaria tenuistipitata var. liui AY673996 ability distributions from these separate replicates were compared Porphyra purpurea U38804 for convergence to the same posterior probabilities across Cryptophytes branches. Majority rule consensus trees of those sampled in Bayes- Guillardia theta AF041468 ian inference analyses yielded probabilities that the clades are Rhodomonas salina EF508371 monophyletic (Lewis, 2001). The trees from the MrBayes analysis Glaucocystophytes were loaded into PAUP* 4.0b10 (Swofford et al., 2001), discarding Cyanophora paradoxa cyanelle U30821 the 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- gies, although often with less resolution and lower posterior quist, 2001), to only include trees after stationarity was estab- probability support for branches. The statistical support of these lished. Posterior probability values (pp) are presented on a relationships is very high as evidenced by the number of branches sample tree from the post-stationarity distribution of Bayesian with posterior probability values greater than 95%. Independent trees in order to demonstrate branch lengths. 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 analy- 3. Results and discussion ses (5 million generations) with 8 chains also converged similarly and fully resolved the consensus topology. Therefore, our results Babesia bovis, an Apicomplexan hemoparasite, is one of the most suggest that (i) the plastids in the Heterokonts Odontella and Tha- prevalent tick-borne pathogens of cattle worldwide. Results from lassiosira originated separately from the Apicomplexans and are the B. bovis genome sequencing project revealed the presence of a likely derived from a red algal lineage (Fig. 1) and (ii) that the api- circular 33 kbp plastid-like genome (Brayton et al., 2007). Although coplast and the Euglenozoan plastids were similarly derived from the function of apicoplasts is not well established, an investigation green plant lineages. into its origin will no doubt provide insight into Apicomplexans’ evo- Overall patterns found here propose that the roots of the Eukary- lution and gene loss in parasites (Keeling, 2004). B. bovis apicoplast otic plastids are in the vicinity of the Glaucocystophyceae and red al- genome together with the recent availability of Theileria apicoplast gae. Strong branch support is found for the placement of the and Helicosporidium plastid sequences allowed us to conduct a com- Apicomplexans with the Euglenozoa and Viridiplantae (Chlorophyta prehensive phylogenetic analysis of apicoplast/plastid genomes and Streptophyta). Further, the Guillardia plastid is found to origi- using nine genes common to all apicoplast/plastid genomes, includ- nate from the red algae, as previously suggested (Hagopian et al., ing rpl14, rpl16, rpoB, rpoC1, rpoC2, rps3, rps11, rps12, and tufA. Anal- 2004) and finally, the Chlorophytes and Streptophytes, as tradition- yses were conducted on deduced amino acid sequence alignments, ally delimited, do not resolve into monophyletic groups (Fig. 1). and these were analyzed individually as well as together in a com- Previous analysis of the phylogenetic position of the Plasmo- bined analysis (Figs. 1 and 2). All of the genes are located on non- dium apicoplast suggested that some data supported a red algal recombining plastids, and therefore, share the same history. These origin, but that a combined analysis of all genes supported a green genes were analyzed for five Cyanobacteria, one Glaucocystophyte, algal origin (Blanchard and Hicks, 1999). The phylogenetic hypoth- two Cryptophyte, four red algae, two Heterokonts, 14 green plants eses presented in that study did not include estimates of branch (five Streptophytes and nine Chlorophytes), two Euglenozoans, one support and, therefore, how strongly one of these topologies was Haptophyceae, one Cercozoa, and five Apicomplexans (Table 1). supported over the other was unclear. Analysis of the individual Although the Apicomplexan, Sarcocystis muris, has been reported genes in our study generally did not provide strong support for to contain an apicoplast, sequences for this organelle are not cur- many of the internal branches of the trees, regardless of the rently available. Cryptosporidium hominis and C. parvum appear to topology found. These results differ from a recent review of plas- have lost their apicoplast and the associated genome, as genes of tid origins (Keeling, 2004) in which it is suggested that both the plastid origin were detected in the nuclear genome but no contigu- Heterokonts and Apicomplexans are derived through secondary ous apicoplast sequence was found in the complete genome (Abra- endosymbiosis from red algae (the chromalveolate hypothesis), hamsen et al., 2004; Wilson et al., 1996; Xu et al., 2004; Zhu et al., whereas the Euglenozoan plastid is derived from a green algal 2000). Gregarina niphandrodes and Selenidium orientale also appear source. Within the Apicomplexans, Babesia and Theileria are sis- to have lost their apicoplast genomes (Toso and Omoto, 2007a,b). ter lineages, as would be expected given their similar plastid It has been suggested that sequences from the apicoplast gen- genome organization, and these taxa together are sister to Plas- ome itself should not be used to determine its phylogenetic posi- modium (Fig. 1). Eimeria and Toxoplasma form a sister clade to tion due to a high AT content that drives long branch attraction this group of three. Furthermore, the parasitic green algae Heli- towards otherwise distantly related lineages (Keeling, 2004). How- cosporidium is strongly placed as sister to the Apicomplexans ever, where amino acid (or DNA) changes have been properly mod- in the combined analysis (Fig. 1), and this relationship shows eled and analyzed under a likelihood framework, these influences support in the rps3, rps11, rps12, rpoB, and rpoC1 individual-gene should be minimized. In addition, we used a covarion model that analyses (Fig. 2). specifically corrects for rate variation across the tree, further min- Results from several studies have been used as a basis for the imizing any potential for a long branch attraction effect from AT chromalveolate hypothesis. For example, red algal origins of the content variation. The suggestion that high AT content in apicop- apicoplast have been suggested using phylogenetic analysis of nu- last genomes improperly forces this lineage towards the green al- clear-encoded, plastid-targeted GAPDH genes (Fast et al., 2001). gae (Morton, 1999) is not supported by the similar AT content in Three aspects of that study should be noted. First, the authors both red and green algae. Last but not least, additional analyses use a complex model of derivation of these nuclear-encoded genes excluding the Euglenozoa plastid genes, which have a highly to explain why the plastid-targeted copies are more closely related biased AT content, were also conducted in our study (data not to eukaryotic cytosolic copies than to plastid copies from plants, shown) and resulted in the same topologies as in Fig. 1. Conse- red or green algae. It is not clear why these results favor red algal quently, we consider the combined data set results in these analy- origins, and while it appears that both the Apicomplexans and ses as the best estimate of relationships of eukaryotic plastids Dinoflagellates have undergone gene replacement, this does not di- without high AT content skewing the overall outcome. rectly address the origins of the plastids to which their nuclear-en- In this study, the analysis of 2826 amino acid (AA) combined- coded gene products are targeted. Second, the branching structure gene with Gblocks matrix resulted in a robust phylogenetic of the phylogeny presented had very low statistical support for hypothesis for apicoplast origins and so this combined data set is most branches, limiting confidence that the presented tree reflects used to represent our consensus hypothesis (Fig. 1). Individual- true branching relationships. Third, since nuclear genomes evolve gene analysis with Gblocks matrices varied in length from 115 to at different rates than those of the plastid (Lynch, 1997; Martin, 854 amino acids (rpl14 – 115 AA; rpl16 – 132 AA; rpoB – 854 AA; 1999), the accuracy of nuclear copies of formerly plastid-encoded rpoC1 – 433 AA; rpoC2 – 509 AA; rps3 – 147 AA; rps11 – 116 AA; genes to represent plastid origins is questionable. rps12 – 121 AA; and tufA – 399 AA). Bayesian analysis of these indi- Gene order and plastid structure have also been used to ad- vidual matrices (Fig. 2A–I) resulted in generally congruent topolo- dress the phylogenetic position of the apicoplast (Blanchard A.O.T. Lau et al. / Experimental Parasitology 123 (2009) 236–243 239

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, Theileria is further complicated by the change from a double- particularly given the extreme levels of gene loss in most of stranded coding structure seen in most plastids to a single- the secondary endosymbionts. Using gene order as a tool for stranded coding arrangement for all genes in the apicoplast gen- comparison of the origin of the apicoplasts from Babesia and omes of these two taxa. 240 A.O.T. Lau et al. / Experimental Parasitology 123 (2009) 236–243

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 apicop- Apicomplexan plastids were not included in this analysis, lasts. Cai et al.’s analysis of rpoB, rpoC1, and rpoC2 genes using branching structure in the Gracilaria study was strongly sup- maximum likelihood and Bayesian inference methods similarly ported and the inferred relationships of photosynthetic taxa provide strong statistical support for this using a smaller sam- were very similar to our results (Hagopian et al., 2004) ple of Apicomplexans and Heterokonts (Cai et al., 2003). A re- (Fig. 1). If Apicomplexan sequences were improperly placed in cent paper on the phylogenetic position of the red algae the current study, we would expect larger perturbations of Gracilaria tenuistipitata var. liui analyzed the relationships the overall tree structure. Several lines of evidence also showed among photosynthetic organisms using 41 plastid protein-cod- the inconsistency of a single origin of the plastid in all Chromi- ing genes (Hagopian et al., 2004). While the Euglenozoa and sta and Alveolata (Bodyl, 2004). A.O.T. Lau et al. / Experimental Parasitology 123 (2009) 236–243 241

Fig. 2 (continued)

The invertebrate pathogen, Helicosporidium, is a green alga of this green alga to the Apicomplexans further strengthens the which has retained a non-photosynthetic plastid (Tartar and Bou- argument that the apicoplast is of green algal origin. Interestingly, cias, 2004; Tartar et al., 2002). This taxon groups very strongly with the association of the apicoplast with a non-photosynthetic green Euglenozoans and Apicomplexans (Fig. 1), and resolves as the sis- alga raises the question of when the plastid became non- ter lineage of the Apicomplexans. The close phylogenetic position photosynthetic. 242 A.O.T. Lau et al. / Experimental Parasitology 123 (2009) 236–243

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 Med- icine, Washington State University.

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