Chromerid genomes reveal the evolutionary path from photosynthetic algae to obligate intracellular parasites

The eukaryotic phylum Apicomplexa encompasses thousands of obligate intracellular parasites of humans and animals with immense socio-economic and health impacts. We sequenced nuclear genomes of Chromera velia and Vitrella brassicaformis, free-living non-parasitic photosynthetic algae closely related to apicomplexans. Proteins from key metabolic pathways and from the endomembrane trafficking systems associated with a free-living lifestyle have been progressively and non-randomly lost during adaptation to parasitism. The free-living ancestor contained a broad repertoire of genes many of which were repurposed for parasitic processes, such as extracellular proteins, components of a motility apparatus, and DNA- and RNA-binding protein families. Based on transcriptome analyses across 36 environmental conditions, Chromera orthologs of apicomplexan invasion-related motility genes were co-regulated with genes encoding the flagellar apparatus, supporting the functional contribution of flagella to the evolution of invasion machinery. This study provides insights into how obligate parasites with diverse life strategies arose from a once free-living phototrophic marine alga. DOI: http://dx.doi.org/10.7554/eLife.06974.001


Introduction
The phylum Apicomplexa is comprised of eukaryotic, unicellular, obligate intracellular parasites, infecting a diverse range of hosts from marine invertebrates, amphibians, reptiles, birds to mammals including humans. More than 5000 species have been described to date, and over 1 million apicomplexan species are estimated to exist (Adl et al., 2007;Pawlowski et al., 2012). Clinically and economically important apicomplexan pathogens, for example, Babesia, Cryptosporidium, Eimeria, Neospora, Theileria, Toxoplasma (Tenter et al., 2000), and the malaria-causing parasite Plasmodium wreak profound negative impacts on animal and human welfare.
Despite their diverse host tropism (Roos, 2005) and life cycle strategies, apicomplexans possess several unifying molecular and cellular features, including the abundance of specific classes of nucleic acid-binding eLife digest Single-celled parasites cause many severe diseases in humans and animals. The apicomplexans form probably the most successful group of these parasites and include the parasites that cause malaria. Apicomplexans infect a broad range of hosts, including humans, reptiles, birds, and insects, and often have complicated life cycles. For example, the malaria-causing parasites spread by moving from humans to female mosquitoes and then back to humans. Despite significant differences amongst apicomplexans, these single-celled parasites also share a number of features that are not seen in other living species. How and when these features arose remains unclear. It is known from previous work that apicomplexans are closely related to singlecelled algae. But unlike apicomplexans, which depend on a host animal to survive, these algae live freely in their environment, often in close association with corals. Woo et al. have now sequenced the genomes of two photosynthetic algae that are thought to be close living relatives of the apicomplexans. These genomes were then compared to each other and to the genomes of other algae and apicomplexans. These comparisons reconfirmed that the two algae that were studied were close relatives of the apicomplexans.
Further analyses suggested that thousands of genes were lost as an ancient free-living algae evolved into the apicomplexan ancestor, and further losses occurred as these early parasites evolved into modern species. The lost genes were typically those that are important for free-living organisms, but are either a hindrance to, or not needed in, a parasitic lifestyle. Some of the ancestor's genes, especially those that coded for the building blocks of flagella (structures which free-living algae use to move around), were repurposed in ways that helped the apicomplexans to invade their hosts. Understanding this repurposing process in greater detail will help to identify key molecules in these deadly parasites that could be targeted by drug treatments. It will also offer answers to one of the most fascinating questions in evolutionary biology: how parasites have evolved from free-living organisms.
We reconstructed the parsimonious gene repertoires for the ancestors of the 26 species, at the nodes of the phylogenetic tree ( Figure 2A; Figure 2-figure supplement 1). We note five key nodes on the evolutionary paths to present-day apicomplexans: the alveolate ancestor; the common ancestor of Apicomplexa and chromerids, termed the proto-apicomplexan ancestor; the apicomplexan ancestor; the ancestor of apicomplexan lineages, for example, coccidia and hematozoa; and extant apicomplexans (Figure 2A). Protein-coding genes from the 26 species were clustered by OrthoMCL  into groups of homologous genes, hereafter defined as orthogroups. We note that an orthogroup could have homologous genes from different species (putative orthologs) or from the same species (putative paralogs arising from gene duplications). Gains or losses of orthogroups are displayed as green or red sections of a pie on the phylogenetic tree in Figure 2A.
Divergence of the proto-apicomplexan ancestor from the alveolate ancestor (Stage I) was accompanied by losses of 1668 and gains of 2197 orthogroups (sum of the two 'pies' in Stage I). Transition of the free-living proto-apicomplexan ancestor to the apicomplexan ancestor (Stage II) is accompanied by many gene losses (3862 orthogroups) but few gains (81 orthogroups) ( Figure 2A). Divergence of coccidians, for example, Toxoplasma gondii, from the apicomplexan ancestor (Stage III) is characterized by modest changes (537 losses; 414 gains), whereas divergence of hematozoans, for example, Plasmodium spp., is marked by drastic losses (1384 losses; 77 gains) ( Figure 2A). Further divergence of apicomplexan taxa beyond Stage III is characterized by modest, lineage-specific gains ( Figure 2A). Functional composition of gained genes at various stages will be discussed in later sections. Paucity of gained genes (81 orthogroups) during Stage II indicates that the genome of the free-living ancestor possessed most of the genes that were present in the common ancestor of apicomplexans and survived in their present-day descendants.
Progressive, lineage-specific losses during apicomplexan evolution Parasite evolution has been associated with genome reduction across several branches of the tree of life (Keeling, 2004;Sakharkar et al., 2004;Morrison et al., 2007). Examples also exist, however, where parasite genomes are not reduced (Pombert et al., 2014) but expanded (Raffaele and Kamoun, 2012), underscoring the fact that the genome reduction process during parasite evolution is not completely understood. We sought to characterize in detail the dynamics of gene loss across apicomplexan evolution, particularly for components of molecular processes that are hallmarks of free-living lifestyle. We performed a systematic analysis of the cellular components involved in: (1) cellular metabolic pathways; (2) the endomembrane trafficking systems, regulating the movement of molecules across intracellular compartments in eukaryotes (Leung et al., 2008); and (3) the flagellum, a highly conserved apparatus for motility in aqueous environment (Silflow and Lefebvre, 2001).  Figure 1-source data 1 for the list of species). Multiple sequence alignments of 101 genes, which have 1:1 orthologs across all species (Figure 1-source data 2) were concatenated to a single matrix of 33,997 aligned amino acids. A maximum likelihood tree was inferred using RAxML with 1000 bootstraps, with Chlamydomonas reinhardtii as an outgroup. All clades are supported with bootstrap values of 100% except one node (*) with 99%, and also with 1.00 posterior probability from a bayesian phylogenetic tree based on PhyloBayes (Lartillot and Philippe, 2004) (CAT-GTR). (B) Lifestyles of the apicomplexan and chromerid species under investigation. '?': uncertainty due to lack of relevant data. DOI: 10.7554/eLife.06974.003 The following source data are available for figure 1: Source data 1. List of 24 species excluding Chomera and Vitrella used in this study and their data sources. DOI: 10.7554/eLife.06974.004 Source data 2. A list of 101 shared orthogroups with a single gene in all of the 26 species, used for the species phylogenetic tree. DOI: 10.7554/eLife.06974.005 . Stages I, II, and III (shown in blue, pink and green, respectively) represent groups of branches from the alveolate ancestor to apicomplexan lineage ancestors. Stage III could not be determined for Cryptosporidium lineage because of sparse taxon sampling. The area of a green or red section in a pie is proportional to the number of gained or lost orthogroups, respectively. (B, C) Overview of metabolic capabilities (B) and endomembrane components (C) in apicomplexan and chromerid ancestors. Gains and losses of enzymes and components were inferred, based on Dollo parsimony (Csuros, 2010). The pie charts are color-coded based on the fraction of enzymes or components present. Additional results from analysis of individual components and enzymes can be found in   The inferred proto-apicomplexan ancestor, like present-day chromerids, possessed complete metabolic pathways for sugar metabolism, assimilation of nitrate and sulfite, and photosynthesis-related functions ( Figure 2B, Figure 2-figure supplement 3, Appendix 2, and Supplementary file 3). Unlike in other photosynthetic algae, both Chromera and Vitrella initiate heme synthesis in the mitochondrion using aminolevulinate synthase (C4 pathway), which thus far has been found only in a few eukaryotic heterotrophs, such as Euglena gracilis, dinoflagellates, and apicomplexans van Dooren et al., 2012;Danne et al., 2013) (Appendix 2 and Supplementary file 4). Both chromerids and apicomplexans encode modular multi-domain fatty acid synthase I (FASI)/polyketide synthase enzymes and single-domain FASII components (Figure 2-figure supplement 2A,B). Treatment of Chromera with a FASII inhibitor triclosan showed decreased production of long chain fatty acids (Figure 2-figure supplement 2C and Appendix 2), suggesting that Chromera synthesizes short-chain saturated fatty acids using the FASI pathway, which are then elongated using the FASII pathway. This was previously demonstrated in Toxoplasma, an apicomplexan that possesses both FASI and FASII (Mazumdar and Striepen, 2007). Likely, the proto-apicomplexan ancestor was a phototrophic alga harboring characteristic metabolic features previously found only in apicomplexan parasites, especially with regard to plastid-associated metabolic functions (see above and other examples in Appendix 2) van Dooren et al., 2012;Danne et al., 2013).
Transition to an apicomplexan ancestor (Stage II) was accompanied by the loss of metabolic processes including photosynthesis and sterol biosynthesis ( Figure 2B and Figure 2-figure supplement 3). The apicomplexan ancestor appeared to possess a significant complement of enzymes in various pathways ( Figure 2B) (Lim and McFadden, 2010). The differentiation of apicomplexan lineages (Stage III) was accompanied by further lineage-specific losses: for example, loss of FASI in Plasmodium spp, loss of FASII in Cryptosporidium spp., which has also lost the apicoplast, and loss of enzymes mediating polyamine biosynthesis in all lineages except Plasmodium ( Figure 2B and Figure 2-figure supplement 3). These support the notion that enzymes involved in cellular metabolism critical for free-living organisms were not completely lost during the transition to the apicomplexan ancestor, but were further lost during subsequent differentiation and host-adaptation of apicomplexan lineages.
The proto-apicomplexan had a nearly complete repertoire of the endomembrane trafficking complexes, and much of this repertoire persisted through to the apicomplexan ancestor (Stage II) (Hager et al., 1999;Klinger et al., 2013a)  Differentiation of apicomplexan lineages (Stage III) was accompanied by lineage-specific losses, for example, loss of the Endosomal Sorting Complex Required for Transport II (ESCRTII) in all lineages except in piroplasms, whereas some components were retained across all lineages, such as the retromer complex components and clathrin, both systems implicated in invasion processes (Pieperhoff et al., 2013;Tomavo et al., 2013)  . These lineage-specific losses have led to diverse, reduced sets of endomembrane trafficking components in present-day apicomplexans (Hager et al., 1999;Klinger et al., 2013a). Some of these components that were present in chromerids were absent in specific apicomplexan lineages as well as in dinoflagellates and ciliates, further clarifying that these losses are independent, lineage-specific events rather than ancient, shared events.
All known components of flagella were present in the proto-apicomplexan ancestor (Figure 2-figure supplement 5A,B). Most of the components were retained in the apicomplexan ancestor (Stage II), but losses occurred as apicomplexan lineages differentiated (Stage III). Components of intraflagellar transport, which are typically essential for assembling flagella, were lost in the other lineages except in coccidians ( Figure 2-figure supplement 5A,B). The basal body proteins, which support an organizing center for microtubules, were lost from piroplasms. Some striated fiber assemblin (SFA) proteins, typically associated with basal body rootlets, were maintained in all apicomplexan lineages including piroplasms ( Figure 2-figure supplement 5A,B,D); their presence has been hailed as evidence that some flagellarproteins are repurposed for new functions in apicomplexans (see below) (Francia et al., 2012).
In summary, one of the major events during apicomplexan evolution is progressive, continued loss of components important for free-living organisms. While Stage II was accompanied by a massive loss of such components including those implicated in photosynthesis, the apicomplexan ancestor still possessed many proteins, which were lost later during differentiation of lineages with diverse life strategies.

Emergent features of apicomplexans
Evolution of present-day apicomplexan parasites was accompanied not only by gene losses as noted above ( Figure 2) but also by gene gains. We sought to determine if genes gained at a particular stage of apicomplexan evolution, as depicted by the gray violin in Figure 3, would be over-represented with those involved in parasitic processes such as intracellular invasion into and egress from host cells. For Plasmodium falciparum and T. gondii, we compiled three classes of protein-coding genes directly or indirectly involved in parasitic processes of apicomplexans based on in silico prediction or information from previous functional studies ('Materials and methods'). Extracellular proteins are secreted by the apicomplexans for various parasitic processes, for example, some of them are targeted to the host cytoplasm, nucleus, and plasma membrane to modulate parasite-host interactions Figure 3. Evolutionary history of Plasmodium falciparum and Toxoplasma gondii genes. Violin plots showing distribution of evolutionary ages of genes (Y-axis: from species-specific (bottom) to deeply conserved (top)) in P. falciparum (A) and T. gondii (B). Evolutionary age of a gene is defined as the earliest node on the evolutionary path of the phylogenetic tree where homolog can be detected ('Materials and methods'). The horizontal thickness of a violin is proportional to the number of genes (gray) or the fraction of genes (yellow) in a functional category (X-axis) out of all with the same evolutionary age. Selected functional sub-categories are overlaid with red, green, or blue violin plots. The maximum width of each violin is scaled to be uniform across categories. Inner boxes in the gray violins indicate inter-quartile ranges and circles indicate medians. Colored shades along the X-axis indicate Stages I-III ( Figure 2). Extracellular proteins include proteins targeted to host cytoplasm, nucleus, and plasma membrane ('exportome') and all other proteins, which are secreted or localized on the parasite surface ('others'). Cytoskeletal proteins include proteins associated with 'actomyosin motor complex' and 'IMC'. All extracellular and cytoskeletal proteins are listed in Figure 3-source data 1, 2. Nucleic acid-binding proteins are predicted in silico based on presence of DNA-binding domains (DBDs) and RNA-binding domains (RBDs). See 'Materials and methods' for details on how these genes are defined and compiled. Domain architectures of representative extracellular proteins in apicomplexans and chromerids are displayed as schematics in     Mundwiler-Pachlatko and Beck, 2013;Bougdour et al., 2014). Cytoskeletal proteins provide structural support to the cell and also the molecular machinery for motility and intracellular invasion Soldati-Favre, 2008). Proteins with DNA-binding domains (DBDs) or RNAbinding domains (RBDs) can regulate various molecular processes of apicomplexan parasites. Indeed, proteins with AP2 (apiAP2) DBD have been shown to act as genetic control switches for diverse apicomplexan processes (Balaji et al., 2005;Campbell et al., 2010;Flueck et al., 2010;Radke et al., 2013;Sinha et al., 2014;Kaneko et al., 2015).
Genes encoding extracellular proteins exported into the host environments were over-represented among those gained after Stage III (Figure 3), suggesting that adaptation to specific hosts was accompanied by expansion of extracellular proteins mediating host-parasite interactions (Templeton et al., 2004a;Anantharaman et al., 2007). Stage III was accompanied by gains of those encoding DBD proteins, mostly apiAP2 proteins (Figure 3 and Figure 3-figure supplement 1A,B), suggesting extensive regulatory changes mediated by apiAP2 proteins during lineage differentiation. We note that losses of other canonical DBD proteins, for example, proteins with HSF_DNA-bind (Pfam: PF00447) domain during transition to apicomplexan ancestor (Stage II) and proteins with Tub (Pfam: PF01167) domain along the piroplasm lineage, contribute to further dominance of apiAP2 among the DBD proteins ( Figure 3-figure supplement 1C). Stage II was accompanied by over-represented gains of various cytoskeletal components, including alveolins, those of the actomyosin complex (e.g., myosins) and glideosome-associated proteins with multiple membrane spans 1 and 3 (GAPM1 and GAPM3), suggesting that the molecular machinery powering gliding motility, which is essential for host cell invasion arose during evolution to apicomplexans (Frenal et al., 2010)  . Chromerid genomes encode many orthologs of apicomplexan cytoskeletal proteins (Appendix 4), including GAPM2, a member of an important protein family for apicomplexan cytoskeletal structure and gliding motility (Bullen et al., 2009), and the IMC sub-compartment protein family (ISP), implicated in establishing apical polarity and coordinating the unique cell cycle of apicomplexans (Poulin et al., 2013) (Figure 2-figure supplement 5E). These data suggest that some components existed in the free-living protoapicomplexan ancestor and were subsequently repurposed for parasitic processes of apicomplexans.
The Chromera and Vitrella genomes encode many proteins that are specific to chromerids yet contain functional domains implicated in molecular processes of apicomplexan parasites. For example, there are chromerid-specific proteins with domain architectures similar to those in apicomplexan extracellular proteins, including those previously implicated in host interactions and described in apicomplexans only (Figure 3-figure supplement 4 and Appendix 5, and Supplementary file 5). Presence of such chromerid proteins implies some commonality in extracellular recognition and crossspecies interactions and this correlates well with the presumed associations with the coral holobiont (Janouškovec et al., 2012(Janouškovec et al., , 2013Cumbo et al., 2013). Importantly, chromerid genomes encode numerous apiAP2 proteins, more abundant than dinoflagellates, suggesting that they have expanded in the proto-apicomplexan ancestor after it split from dinoflagellates ( Figure 3-figure supplement 1D). Many of the chromerid apiAP2 proteins belong to putative paralogous clusters, suggesting that their expansion was driven by gene duplication (Figure 3-figure supplement 1D; Appendix 6). Only a small subset of the apiAP2 proteins are shared across apicomplexans, suggesting that the large apiAP2 complement in the proto-apicomplexan ancestor has diversified independently in descending lineages ( Figure 3-figure supplement 1A).
In summary, genes encoding critical components of the parasitic lifestyle of apicomplexans were gained at different stages of apicomplexan evolution, some implying subsequent specialization to particular host niches, but others suggesting early adaptations before committing to parasitic lifestyle. This is evident by chromerid orthologs of many such proteins, for example, RAP proteins and specialized cytoskeletal components. Further, chromerid genomes encode chromerid-specific proteins that are not detected as orthologs of apicomplexan proteins but still have functional domains implicated in parasitic processes in apicomplexans. Together, these data imply that a molecular transition had occurred in free-living ancestors of apicomplexans, providing a foundation for host-parasite interactions and further adaptation.

Conserved gene expression programs in the proto-apicomplexan ancestor
Chromera and Vitrella genomes allowed us to reconstruct the gene content of the free-living ancestor of apicomplexans. To infer their putative functions using genome-wide gene expression information (Hu et al., 2010), we cultured Chromera under 36 different combinations of temperatures, iron and salt concentrations, and generated their gene expression profiles by RNA-seq (Box et al., 2005). In addition, we have obtained a publicly available growth perturbation data set for P. falciparum (Hu et al., 2010). There were 1918 orthogroups shared between the two species. We identified pairs of orthogroups that are co-expressed, that is, showing similar expression patterns across the various conditions, in both species ('Materials and methods') ( Figure 4-figure supplement 1A). Such an orthogroup pair, that is, those with conserved co-expression between the two species, would include candidate genes that have been co-regulated together during apicomplexan evolution, from the freeliving ancestor to present-day parasites due to conserved functions. This approach, successfully utilized by several studies in the past (Stuart et al., 2003;Mutwil et al., 2011;Gerstein et al., 2014), led to the following two observations in this study.
Many RAP genes appeared during Stage I and have been conserved across the descending phyla ( Figure 3 and Figure 3-figure supplement 3), but their precise cellular roles are unknown. For 11 out of 12 orthogroups with RAP domains, co-expressed orthogroups overlapped significantly (Fisher's exact test, p < 0.05) between P. falciparum and Chromera, suggesting involvement of RAP proteins in cellular processes evolutionarily conserved across apicomplexans and chromerids ( Figure 4A). RAP and their co-expressed orthogroups encode proteins with putative mitochondrial import signals more often than expected by chance in Chromera and P. falciparum (Fisher's exact test, p < 0.05) ( Figure 4B), and also in other apicomplexans and chromerids (Figure 4-figure supplement 1B). We have randomly chosen three Toxoplasma RAP genes with predicted mitochondrial localization signals (Supplementary file 6) and confirmed experimentally by 3′ endogenous gene-tagging with reporter epitopes that all three are localized to the organelle ( Figure 4C). Some of the orthogroups coexpressed with orthogroups containing RAP domains encode protein products predicted to be metabolic enzymes, implying possible involvement of RAPs in mitochondrial metabolism (Figure 4-figure supplement 1C). Consistent with this, the Cryptosporidium lineage that has a highly reduced mitochondrion lacking both the genome and most canonical metabolic pathways Xu et al., 2004) is the only apicomplexan group to have also lost its RAP repertoire ( Figure 4-figure supplement 1D). Loss of RAPs along with a set of mitochondrial functions in this lineage is consistent with a mitochondrial role for RAPs. We speculate that the freeliving proto-apicomplexan ancestor possessed within its mitochondrion a regulatory process mediated by RNA-binding activities of the RAP proteins, which has been retained by the extant apicomplexans and chromerids.
As discussed earlier, the proto-apicomplexan ancestor appears to have possessed genes implicated in invasion processes of present-day apicomplexans ( Figure 3). Among the 1918 orthogroups, we identified 80 orthogroups comprising genes functionally annotated as implicated in invasion processes. The frequency of co-expression amongst them in the free-living Chromera was significantly higher than expected by chance (p < 0.0005), suggesting pre-existing functional relationships before transitioning to parasites ( Figure 4D). We identified several modules or groups of co-expressed orthogroups ( Figure 4E). In one of the co-expression modules (numbered 1 in Figures  4E), 9 out of 10 orthogroups are co-expressed with a gene encoding SFA (Cvel_872), a key protein for organizing the basal bodies of the flagellar apparatus in algae and the apical complexes in apicomplexans (Kawase et al., 2007;Francia et al., 2012) ( Figure 4F). We note that SFAs are the only flagellar components found in all apicomplexans tested (Figure 2-figure supplement 5A). Also in this module, for 9 out of 10 orthogroups, their co-expressed orthogroups in Chromera overlapped significantly with those in P. falciparum (Fisher's exact test, p < 0.05), indicating that their regulatory programs have been evolutionarily conserved ( Figure 4G). This module include various types of genes implicated in host cell invasion processes of apicomplexans such as genes encoding rhoptry protein ROP9, apical sushi protein ASP, and gliding motility components GAP40 and GAPM2. The apical complex has been postulated to have emerged from the flagellar apparatus and associated cellular transport systems in free-living algae, based on ultrastructural evidence (Okamoto and Keeling, 2014;Portman et al., 2014). These results suggest that, in the free-living ancestor, some of the genes programs for all orthogroups or those with RAP domains. X-axis: 'All' (all orthogroups excluding RAP); 'RAP' (orthogroups with RAP domains). Y-axis: log-transformed odds-ratio, representing, for each orthogroup, the degree of overlap between its co-expressed orthogroups in Chromera and those in P. falciparum. (B) Bar chart showing the fraction of orthogroups (Y-axis) predicted to be targeted to mitochondria in both species ('Materials and methods'). The number of genes are displayed below each bar. X-axis: 'All' (all orthogroups excluding the other two categories); 'Coexpr' (orthogroups co-expressed with RAP in both species); 'RAP' (orthogroups with RAP domains). The fractions in 'Coexpr' and 'RAP' groups were compared against the fraction in 'All', and p-values based Fisher's exact test are displayed above the bar. Files deposited in European Nucleotide Archive are listed in Figure 4-source data 1 with corresponding conditions. (C) Sub-cellular Figure 4. continued on next page implicated in the invasion process of present-day apicomplexans were functionally associated with those implicated in flagellar motility, providing the much-needed genetic evidence for the postulate. We speculate that a group of functionally related proteins associated with the flagellar apparatus was repurposed as a module of the apical complex and became a foundation for the invasion machinery.

Conclusion
Analysis of Chromera and Vitrella genomes has enabled insights into how apicomplexan parasites have evolved from free-living ancestors. The transition to parasitism was accompanied by massive genomic loss that continued as its descendants became specialized intracellular parasites infecting diverse hosts. The genome of free-living photosynthetic ancestors encodes many component proteins previously assumed to be restricted to the parasitic apicomplexan lineages. Such pre-existing components, including those of what would later become part of the invasion machinery, were coopted during evolution to facilitate a successful parasitic lifestyle in multiple hosts. The genome of the proto-apicomplexan ancestor served as a molecular blueprint for evolution of the most successful group of eukaryotic parasites known to date.

Data access
Sequencing data have been deposited in the European Bioinformatics Institute under the European Nucleotide Archive (ENA) sample accession number ERP006228 for C. velia and ERP006229 for V. brassicaformis for all DNA-and RNA-seq experiments. The assembly and the annotations were submitted under accession numbers CDMZ01000001-CDMZ01005953 for C. velia and CDMY01000001-CDMY01001064 for V. brassicaformis. Some of the Vitrella DNA-seq experiments were done at Broad Institute and are deposited at Short Read Archive under accession numbers SRX152523 and SRX152525. The annotations and assemblies can be viewed and queried in EupathDB (http://cryptodb.org/cryptodb/).

DNA preparation and sequencing
Genomic DNA of C. velia CCMP2878 (subsequently referred to as Chromera) and V. brassicaformis CCMP3155 (subsequently referred to as Vitrella) was extracted and then sheared into short fragment localization of RAP proteins encoded by TGME49_237010, TGME49_269830, and TGME49_289200 was tested in T. gondii by 3′ tagging of the endogenous genes with the coding sequence for the hemagglutinin epitope, together with a mitochondrial marker Tom40. See Supplementary file 6 for details of the localization predictions. (D) Distributions of Spearman's rank correlation coefficients of gene expression between all possible pairs from the 80 orthogroups implicated in invasion processes in apicomplexans (black outline) were compared against those from 80 randomly selected ones (histogram). The p value indicates statistical significance of the difference based on 10,000 random samplings. The 80 orthogroups and corresponding genes in Chromera and P. falciparum are listed in Figure 4-source data 2. (E) Heatmap showing a matrix of correlation coefficients amongst the 80 orthogroups. Based on a hierarchical clustering, we classified them into six co-expression modules, labeled as numeral 1-6. (F) Heatmap showing correlation coefficients with striated fiber assemblin (SFA) (Cvel_872). The color scheme is the same as in (E). (G) Heatmap indicating statistical significance of conserved transcriptional program, that is, the oddsratio as defined in (A) (Fisher's exact test, p < 0.05 (gray); p < 0.005 (black)). DOI: 10.7554/eLife.06974.022 The following source data and figure supplement are available for figure 4: Source data 1. RNA-seq libraries of Chromera velia under various growth conditions. DOI: 10.7554/eLife.06974.023 Source data 2. List of genes implicated in invasion processes in apicomplexans. DOI: 10.7554/eLife.06974.024 Source data 3. Evolutionary conservation of 12 orthogroups with RAP domains (for 'RAP' category in Figure 4A). DOI: 10.7554/eLife.06974.034 size libraries (300-500 base pair (bp)) and large fragment size libraries (3-8 kbp fragments) by focused-ultrasonication (Covaris Inc., Woburn, USA). The last 3-8 kb libraries were prepared following Nextera mate pair protocol, following manufacturer's instructions. We used three different methods to generate the library: the Illumina (Illumina, San Diego, CA) TruSeq DNA protocol LT Sample Prep Kit (catalog no. #FC-121-2001), an amplification-free method (Kozarewa et al., 2009) (TruSeq DNA PCR-Free LT Sample Preparation Kit catalog no. #FC-121-3001) and the Illumina Nextera Mate Pair Sample Preparation Kit (catalog no. #FC-132-1001). The libraries were sequenced on an Illumina HiSeq2000 platform following the manufacturers standard cluster generation and sequencing protocols (Bentley et al., 2008;Quail et al., 2012). Image analysis, base-calling, and quality filtering were processed by Illumina software.

RNA preparation and sequencing
For isolation of RNAs, Chromera and Vitrella were grown under standard culture conditions (Oborník et al., 2012). Total RNA was extracted from the cells using TRIzol. The polyA+ RNA fraction was selected using oligo(dT) beads, and RNA-seq libraries were prepared using TruSeq RNA Sample Prep kit (catalog no. FC-122-1001). Strand-specific RNA-seq libraries were prepared using TruSeq Stranded mRNA LT Sample Prep Kit (catalog no. RS-122-2101) and sequenced as paired-end (2 x 100 bp) reads on a HiSeq2000 platform.
We performed additional RNA sequencing of Chromera subject to various environmental perturbations, to construct a global gene expression network based on transcriptomes under various perturbation conditions during in vitro growth. Chromera cultures were exposed to a combination of stresses (Figure 4-figure supplement 1C). First, six different media were prepared from the combinations of salt concentration (16.7 g/l, 33.3 g/l, 66.6 g/l) and iron deficiency by chelation (Sutak et al., 2010). After seeding, the cultures were maintained in the normal temperature and light condition for eleven days . After randomization, the cultures were incubated at 26˚C, 37˚C, or 14˚C for 0 (control), 0.5, or 2 hr. There were two biological replicates of each, in total 66 flasks of the cultures. Then, the cultures were processed with centrifugation at 3500 RPM for 15 min at 4˚C to precipitate the cells. Total RNA was extracted from the 66 cultures after the treatments using Norgen RNA Extraction kit based on manufacturer's protocol (Norgen Biotek Corporation, Canada). RNA quality was assessed using Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). RNA concentration was determined with a Qubit (Invitrogen, Carlsbad, CA). Strand-specific RNA-seq libraries were prepared from extracted high-quality RNAs (RIN ≥8.0 as measured on an Agilent Bioanalyser 2100) using the Illumina TrueSeq LT stranded RNA sample kit according to manufacturer's instructions. Prior to cluster generation, concentration and size of libraries were assayed using the Agilent DNA1000 kit. Libraries from all samples were sequenced as single-end (1 x 50 bp) reads on the Illumina HiSeq 2000. The RNA-seq reads were aligned to the reference genome using tophat (version 2.0.8, default parameters) and cufflinks (version 2-1.0.2, default parameters) (Trapnell et al., 2012). The FPKM values were log2 normalized with an offset of 1 and were further corrected for different distributions across the samples using the quantile normalization method (Bolstad et al., 2003).

Genome assembly
For Vitrella, the reads were corrected and assembled followed by several base correction, scaffolding and gap filling steps as briefly described below. As first step, the short insert libraries were corrected with SGA (Simpson and Durbin, 2012) (version 0.9.19). The corrected reads were assembled with velvet (Zerbino and Birney, 2008) (version 1.2.08). Iterating through different parameter settings, we choose a k-mer of 75 bp as the best parameter set. The resulting scaffolds (larger than 1 kb) were further scaffolded with SSPACE (Boetzer et al., 2011) using first the Illumina library (insert = 550 bp) and larger insert (1 kb) Illumina library reads. Sequencing gaps were closed with Gapfiller (Boetzer and Pirovano, 2012) (version 1.1.1) with two iterations, using the bowtie mapping option and PCR-Free libraries. Base pair call errors were corrected in three iterations of ICORN , using the amplification-free library. Furthermore, sequencing gaps were closed, using IMAGE (Tsai et al., 2010) with the amplification-free library. The assembly was quality-controlled using REAPR (Hunt et al., 2013), breaking the contigs at possible miss-assemblies, using the mate pair libraries. This was followed by another scaffolding step. We systematically removed 620 scaffolds containing 25.65 Mb representing the bacterial contamination. The Vitrella CCMP3155 assembly contains 72.7 Mb (including 931,689 N's) in 1064 scaffolds (ENA accession numbers CDMY01000001-CDMY01001064). The scaffolds were constructed from 4177 contigs.
For Chromera, the assembly pipeline and the algorithms used were the same as Vitrella, but due to the larger size, higher amount of low-complexity regions, and difficulties in generating high-quality large insert size libraries, additional steps were included to the assembly process. First, the reads of the PCR-Free library were corrected with SGA (Simpson and Durbin, 2012) and then assembled with velvet and using a k-mer of 71 (version August 2011). Next, the contigs were scaffolded, gapfilled, and corrected with ICORN, as described earlier. We mapped the reads of all large insert size libraries using SMALT (ftp://ftp.sanger.ac.uk/pub/resources/software/smalt/). We excluded scaffolds smaller than 1 kb. Different iterations with SSPACE were undertaken and the assembly was quality-checked with REAPR. After scaffolding, gapfiller and IMAGE were run as above, followed by ICORN. The 1725 scaffolds (spanning 16.02 Mb) representing bacterial contamination were removed. The final assembly of Chromera CCMP2878 contains 193.66 Mb (including 582,995 N's) in 5953 scaffolds (ENA accession numbers CDMZ01000001-CDMZ01005953). The scaffolds are constructed from 13,987 contigs.

Gene prediction
We used Augustus (Stanke et al., 2006) (version 2.5.5) for gene prediction. We manually curated 716 and 245 gene models for Chromera and Vitrella, respectively, using BLAST similarity-based approaches, and we also generated automated gene models using Cufflinks (Trapnell et al., 2012) from RNA-seq data sets, in order to use them as a 'training gene model set' for Augustus prediction. The strand-specific RNA-Seq, mapped with TopHat2 (Kim et al., 2013), was used as evidence in Augustus for intron evidence.
In summary, from the Chromera and Vitrella genome, we ab initio predicted 30,478 and 23,503 protein-coding genes, respectively, of which 18,829 and 18,240 were detected as being expressed from RNA-seq evidence as poly A+ transcripts (Supplementary file 1). Excluding putative TEs, 26,112 and 22,817 genes were predicted as protein-coding genes in Chromera and Vitrella. We annotated partial genes, when a gene probably spans more than one scaffold, located at the borders of a scaffold. We demarcated and annotated as pseudo genes if they contain in frame stop codons. We flagged gene models as transposon elements, if they overlap with the predicted TE regions and had no more than three and two intron for Chromera and Vitrella, respectively. To annotate untranslated regions (UTRs) of the predicted protein-coding genes, we used CRAIG (Bernal et al., 2007) with default parameters with mapping of the RNA-Seq data as computed by GSNAP (Wu and Nacu, 2010) (version 2013-08-19, default parameters). The annotation of both genomes has the ENA accession numbers CDMZ01000001-CDMZ01005953 and CDMY01000001-CDMY01001064 and is also available in EuPathDB (Aurrecoechea et al., 2013).
We collected several categories of genes implicated in parasitic processes in apicomplexans for two archetypal apicomplexan parasites, Toxoplasma and Plasmodium. We primarily obtained annotations from PlasmoDB (Bahl et al., 2003) and ToxoDB (Gajria et al., 2008). Information for sub-cellular localization of genes is obtained from GeneDB (Logan-Klumpler et al., 2012) and ApiLoc, a database of published protein sub-cellular localization for apicomplexan species (http://apiloc.biochem.unimelb.edu.au/apiloc/apiloc). Some putative parasite genes were inferred based on orthology by OrthoMCL clustering  with closely related species with results from functional studies. We performed exhaustive literature searches to manually curate individual genes, to define rules for in silico searches across the proteomes of this study, and to categorize the identified genes based on their localization and function. The categories of parasite genes are defined as follows.

Cytoskeleton
The cytoskeleton of an organism provides the necessary structural framework for the maintenance of cell shape and integrity. We compiled two groups of cytoskeletal proteins, IMC associated proteins and actomyosin complex. First, IMC associated proteins, comprises alveolin proteins, a membrane occupation and recognition nexus protein (MORN), which associate with IMC and spindle poles and are indispensible for asexual and sexual development (Ferguson et al., 2008). IMC sub-compartment proteins (ISPs) are critical for establishing apical polarity in the parasite (Poulin et al., 2013). Second, components of actomyosin motor complex, which powers the characteristic gliding motility (Soldati-Favre, 2008), comprises actin, myosin, tubulin, gliding associated proteins (GAPs), aldolase, and various actin-regulatory proteins, which will assist actin in the process of quick polymerizationdepolymerization cycles between F-actin and G-actin during this process. Examples of actinregulatory proteins are Arp2/3 complex and formins (FH2) for nucleation; F-actin capping for filament regulation; coronin for cross-linking/bundling and profilin, CAP, cofilin/ADF and gelsolin for monomer treadmilling .

Extracellular proteins
Extracellular proteins are defined as parasite proteins, which are localized either on the surface or secreted off the parasite. They are released in a concerted manner to ensure successful adhesion to the surface, entry into the host cell, multiplication, and escape. Extracellular proteins can be categorized as (1) 'exportome' are proteins translocated to the host cytoplasm, membranes, and nucleus crossing the boundary membrane parasitophorous vacuole (PV); and (2) 'others', which stay on the parasite surface or released from the parasite, but not into the host intracellular space. The exportome genes are released mostly from the parasite's secretary organelles such as rhoptries and dense granules (Ravindran and Boothroyd, 2008;Treeck et al., 2011;Mundwiler-Pachlatko and Beck, 2013;Bougdour et al., 2014). Some of these genes possess host targeting or also known as the Plasmodium export element (PEXEL). Many PEXEL-negative proteins have been identified too (Hsiao et al., 2013;Mundwiler-Pachlatko and Beck, 2013). These genes are sorted and targeted through a specialized structure known as Maurer's cleft formed in the host cytoplasm (Mundwiler-Pachlatko and Beck, 2013). These genes are mostly kinases, proteases, and surface molecules, which modulate the host and hijack the host machinery in favor of parasitic growth and host immune evasion (Treeck et al., 2011;Li et al., 2012;Bougdour et al., 2014). The 'other' extracellular proteins consist of surface antigens (e.g., MSPs), SERAs, TRAPs, AMA-1, microneme proteins, ROPs and RONs etc.

TEs
Repeat annotation was done by using the REPET pipeline (Flutre et al., 2011) and LTR finder (Xu and Wang, 2007). The overall pipeline comprises of two steps: de novo detection and classification. In the first step, the scaffolds are split into smaller batches (∼1000 batches of 200 kb each). These genomic fragments were aligned against each other to detect the HSPs (High-scoring pairs) using BLASTER (Quesneville et al., 2003). HSPs are then clustered using a combination of three methods such as GROUPER (Quesneville et al., 2003), RECON (Bao and Eddy, 2002), and PILER (Edgar and Myers, 2005). Structure-based LTR retrotransposons (RTs) detection tools such as LTRharvest (Ellinghaus et al., 2008) and LTR finder, which are based on 100-1000 bp long terminal repeats with a 1 kb-15 kb separation and target site duplication site at vicinity of 60 bp to the two terminal repeats. These LTRs detected are clustered using BlastClust. Multiple sequence alignment of each cluster was performed using MUSCLE (Edgar, 2004). Each cluster aligned was searched against Repbase (Jurka et al., 2005) using BLASTER (Quesneville et al., 2003) and HMMER (Johnson et al., 2010). A consensus feature was detected for each aligned cluster. Further PASTEC (Flutre et al., 2011), which is based on the Wicker classification, was used for consensus classification.
The repeats were annotated as follows. The genomic chunks were randomized and HSPs were detected using BLASTER (Quesneville et al., 2003), CENSOR (Jurka et al., 1996), and RepeatMasker (Tempel, 2012). These HSPs were filtered and combined. Again, full-length genomic scaffolds were compared to Repbase using MATCHER. Satellite and simple repeats were detected using the mreps (Kolpakov et al., 2003), TRF (Benson, 1999), RMSSR (RepeatMasker). Finally, a long-join procedure was followed to combine the nested repeats. The whole annotation was exported to a genomebrowser readable GFF3 file.

Clustering homologous genes
OrthoMCL 2.0  was used with a default inflation parameter (I = 1.5) (Chen et al., 2006) to generate groups of homologous genes (defined as orthogroups), which could have homologs from different species (putative orthologs) or from the same species (putative paralogs from gene duplications). For some genes of high interest, we manually inspected the alignments of the protein sequences within the orthogroup, which were done with MAFFT (Katoh and Standley, 2013). We assigned Pfam domains to an orthogroup if more than half of the genes in an orthogroup were assigned the Pfam domains.

Sub-cellular localization prediction
There are several tools available for a general eukaryotic sub-cellular localization prediction (Du et al., 2011), but they are not applicable to alveolates due to its unique chloroplast membrane arising from secondary endosymbiosis. Therefore, HECTAR (Gschloessl et al., 2008), which was developed for the bipartite sub-cellular prediction, was used. There is no stand-alone version of HECTAR, and the online version allows only one sequence at a time. We implemented a modified HECTAR algorithm as a PERL script for batch prediction of the whole proteomes. Each protein sequence was predicted for signal sequence using SignalP 3.0 (Bendtsen et al., 2004), the signal sequence is cleaved, and the remaining amino acid sequence was used as input for the transit peptide prediction by TargetP (Emanuelsson et al., 2000). Sequences with both signal peptide and the transit peptide (either chloroplast or mitochondria) are predicted to be in the chloroplast. Sequences without the signal peptide but with the transit peptide (either chloroplast or mitochondria) are predicted to be in mitochondria. Sequences with signal peptide, without transit peptide, and predicted by TargetP to be secretory are classified as secretory proteins.
For the RAP proteins, we tested the validity of our sub-cellular localization prediction in two ways. First, we compared our in-house algorithm with other published tools: TargetP (Emanuelsson et al., 2000), MitoProt2 (Claros and Vincens, 1996), iPSORT (Bannai et al., 2002), and PredSL (Petsalaki et al., 2006) (Supplementary file 6, only mitochondrial prediction is shown). We found that our mitochondrial prediction for RAP genes is in concordance with other methods. Second, we experimentally verified mitochondrial localization in T. gondii by 3′ tagging of the endogenous genes with the coding sequence for the hemagglutinin epitope for three RAP proteins that were predicted to target to mitochondria with high probability.

Statistical analysis
A statistical environment software R was used for most of the analyses and generating parts of figures. An R package vioplot was used to generate the violin plot (Hintze and Nelson, 1998). A ward algorithm on the distance matrix based on (1-correlation coefficients) in an R function hclust was used for all hierarchical clustering of gene expression patterns unless noted otherwise.
We generated a phylogenetic species tree using a data set composed of 101 one-to-one orthologs across the 26 species (see Figure 1-source data 1 for gene IDs). Amino acid sequences were aligned using MAFFT (Katoh and Standley, 2013), highly variable sites were edited by trimAL (Capella-Gutierrez et al., 2009) and after manual inspection. The resulting alignment of 33,997 amino acid positions was used to construct trees by a maximum likelihood (ML) method and Bayesian inference. The ML tree was computed using RAxML 8.1.16 by gamma corrected LG4X model (Stamatakis, 2014;Le et al., 2012). Robustness of the tree was estimated by bootstrap analysis in 1000 replicates. Bayesian tree was constructed by PhyloBayes (Lartillot and Philippe, 2004) using two-infinite mixture model CAT-GTR as implemented in PhyloBayes 3.3f. Two independent chains were run until they converged (i.e., maximum observed discrepancy was lower than 0.2), and the effective number of model parameters was at least 100 after the first 1/5 generation was omitted from topology and posterior probability inference. All clades in the tree were supported with posterior probability 1.00 and 100% bootstraps, except for one node, which representing the common ancestor of human Plasmodium spp. was supported by 99% bootstrap.
We performed the gene gain and loss analysis based on Dollo parsimony using Count software (Csuros, 2010). This approach allows reconstructing gene contents at observed species and at hypothetical ancestors, and gene gains and losses at branching points. The Dollo parsimony strictly prohibits multiple gains of genes. To test for validity of this assumption, we repeated analyses based on parsimony settings allowing multiple gene gains or on a phylogenetic birth-and-death model (Csuros, 2010) and reached the same conclusion (Figure 2-figure supplement 1). We have also repeated the analysis using Wagner's parsimony, allowing multiple gains per tree with gain penalty of 2 or greater, and obtained similar results (data not shown). For the analysis of metabolic enzymes, endomembrane trafficking system components, and flagellar apparatus components, the ancestral presence was inferred based on Dollo parsimony from the presence of components in the observed species. For the endomembrane trafficking component analysis, we assumed that the last common ancestor had a complete repertoire of the components.
We have inferred the evolutionary age of P. falciparum and T. gondii genes as the early node on the phylogenetic tree where the most distant species have genes with significant sequence homology (reciprocal BLASTP E value <10 −10 and clustering with OrthoMCL).

Comparison of gene expression network between Chromera velia and Plasmodium falciparum
We studied if orthologs of Chromera and P. falciparum show similar gene expression changes to physiologically equivalent growth conditions. Identifying equivalent conditions is difficult as the two species have completely different lifestyles and live in different environments. Instead, we tested if a given gene and its ortholog would show correlated expression patterns with the same set of genes (and orthologs), allowing a way to compare gene expression behavior measured under different conditions. To uncover gene-to-gene co-expression relationships, the organisms from whom transcriptomes are sampled must be exposed to various growth conditions. This approach has been successfully used in other eukaryotes (Stuart et al., 2003;Hu et al., 2010;Mutwil et al., 2011). For Chromera, we generated RNA-seq-based transcriptome under combinations of varying salt concentrations, iron concentrations, and temperature changes, resulting in 36 unique combinations (see 'Materials and methods' and Figure 4-figure supplement 1C). For P. falciparum, we obtained previously published microarray-based gene expression data sets of 144 unique conditions from 23 time series, representing stresses from various growth-inhibiting compounds (Hu et al., 2010). It has been shown that gene expression data generated using different molecular platforms are reproducible and accurate enough for cross-platform comparisons (Woo et al., 2004). Based on each data set, we calculated Spearman correlation coefficients rho between all possible pairs from the 1918 orthogroups shared between Chromera and P. falciparum (1918 × 1918 matrix). We also calculated a 1918 × 1918 weighted adjacency matrix using CLR algorithm (Faith et al., 2007) as implemented in an R package minet (with parameters of method = 'clr', estimator = 'mi.shrink', and disc = 'equalfreq') (Meyer et al., 2008). Expression level of multiple genes in a given orthogroup was averaged. To rule out any potential systematic biases associated with averaging expression levels of homologous, yet distinct genes, we repeated some of the analyses with 1560 orthogroups that have one-to-one orthologs between the two species and reached the same conclusions (data not shown). A pair of genes (or orthogroup) were determined as co-expressed if the Spearman's correlation coefficient rho is greater than 0.3 and if the value from the weighted adjacency matrix of the network is greater than 0.01. We calculated an odds-ratio to measure the extent of conservation of co-expressed genes: (# of genes co-expressed in both species) × (# of genes co-expressed in none of the species)/([# of genes co-expressed in P. falciparum only] × [# of genes co-expressed in C. velia only]), and Fisher's exact test was used to assess the statistical significance. For calculation of the odds-ratios, co-expression was determined based on correlation coefficient to minimize count granularity in the two-by-two table.

Acknowledgements
We thank the KAUST Bioscience Core Laboratory personnel for sequencing specific Illumina libraries used in this project, KAUST Computational Bioscience Research Center for providing computing resources, Gordon Langsley (Institut Cochin, Inserm U1016, Paris) and Anthony Holder (The Francis Crick Institute, London) for comments on the manuscript draft. AV and CB thank Florian Maumus for advice regarding TE annotation. The primary funding for this work was provided by KAUST award FIC/ 2010/09 to JL, MO, and AP. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.   Cvel_967). (e) Cysteine-rich domain found in Cryptosporidium oocyst wall proteins (COWP) and in coccidians. (f) Archaeal protease-type repeats first described in the Cryptosporidium predicted EC protein, cgd7_4560. The domain was previously described as 'A small domain with characteristically spaced cysteine residues that is fused to a papain-like protease domain in the secreted protein AF1946 from Archaeoglobus fulgidus (Templeton et al., 2004a)'. (g) The domain was previously described as 'Domain typically with 6 cysteines, seen thus far mainly in animals with a few occurrences in plants. It is found in the sea anemone toxin metridin and fused to animal metal proteases, plant prolyl hydroxylases and is vastly expanded in the genome of Caenorhabditis elegans (Templeton et al., 2004a)'. (h) The domain was previously described as 'β-strand rich domain, predicted to form a β-sandwich structure that is found in bacterial secreted levanases and glucosidases (Templeton et al., 2004a)

Major datasets
The following datasets were generated: Author (  The statistics of the genome assembly and annotation are shown in Supplementary file 1. There was bacterial contamination in 20% and 80% of the sequence reads in Chromera and Vitrella, respectively. There was a high amount of low-complexity DNA sequence repeats and TEs in Chromera (Supplementary file 1). By various bioinformatics methods ('Materials and methods'), we generated assemblies containing 5953 and 1064 scaffolds for Chromera and Vitrella, respectively. The total number of predicted genes differed between Chromera and Vitrella primarily due to significant differences in TE gene content between the two chromerids but the number of expressed genes was similar (Supplementary file 1).
We examined how genomes of the chromerids and other species were organized (Supplementary file 1). The median gene length is roughly the same between the two chromerids. The number of introns in a given gene was similar between the chromerids, although the size of introns was larger in Chromera than in Vitrella (Supplementary file 1). Compared to these chromerids, the number of introns in Apicomplexa was drastically less, raising the possibility that introns were compacted and reduced during apicomplexan evolution, which would need to be confirmed with further detailed investigation. For many genes (13,912 and 17,569 respectively for Chromera and Vitrella), we were able to assign 5′ and 3′ UTRs, using strand-specific transcriptome (RNA-seq) data sets. The distance between the protein-coding genes in Vitrella was short (median 92 base-pairs (bp)), indicating compactness of its genome. On the other hand, such distance was longer in Chromera (median 989 bp). Determining whether the common ancestor of chromerids had a compact genome or not would require analysis of genomes from more closely related species. There are three possible orientations by closely spaced neighboring genes can be clustered, that is, those with short intergenic spaces between the gene boundaries: tandem, head-to-head, or tail-to-tail. In both Chromera and Vitrella genomes, closely spaced (<1000 bp) genes were in head-to-head orientation more often than expected by chance (data not shown). It was previously shown that many neighboring genes in head-to-head clusters showed correlated expressions across various conditions; however, most of the co-expressions were modest; instead, head-to-head clustering is a major mechanism for stabilizing transcription of genes in fundamental cellular processes rather than for co-regulating the two genes (Woo and Li, 2011;Russell et al., 2013). Head-to-head clustering probably provided evolutionary and regulatory stability to genes involved in fundamental cellular processes. Other related species had different gene orientations, for example, the dinoflagellate Symbiodinium microtinum has tandem clusters driven by tandem gene duplication (Shoguchi et al., 2013). Given the dynamic nature of genome organization, we propose that different groups of species evolved different strategies for genome organization (Woo and Li, 2011).
Repetitive sequences constitute a significant proportion of eukaryotic genomes (Fedoroff, 2012). Thus, they play a significant role in evolution of host genomes. Systematic TE clustering, classification, and annotation were performed on 1064 Vitrella scaffolds (72.7 Mb genome-72,700,666 bp) and 5953 Chromera scaffolds 664,168 bp) Chromera. In both species, Class I elements (Tempel, 2012) make up a larger proportion of the genome than Class II elements (Tempel, 2012) (Supplementary file 2). The RT domain variation shows that Eimeria tenella TEs grouped separately and are not related to chromerid TEs (Supplementary file 2), suggesting gains of TEs in E. tenella (Reid et al., 2014) independently from chromerids. Vitrella forms a separate clade in the phylogenetic analysis of the RT domains. Metabolism.

Materials and methods
Reconstructing global metabolic map based on KEGG Global metabolic pathways were mapped to KEGG metabolic pathways for the predicted protein-coding gene sets for the 26 species. KEGG ortholog (KO) assignments for the respective proteomes were made using the KO identification tools available on the KEGG website (http://genome.jp/tools/kaas) (Moriya et al., 2007;Kanehisa et al., 2014), and then the assigned KO numbers were used to identify and map metabolic pathways using the 'Search and color pathway' tool available on the KEGG site (http://genome.jp/kegg/tool/map_path-way2.html). The output of this mapping exercise was then manually inspected to compile the set of enzymes present in all major metabolic pathways (Figure 2-source data 1).
As KEGG is very strict in mapping orthologs and assigning KO numbers (so as to minimize false positives), we found numerous pathway holes (missing enzymes), many of which were readily apparent as false negatives. In order to resolve this, we then resorted to identifying orthologs from OrthoMCL-DB for all the genomes compared here (Chen et al., 2006). The resulting ortholog assignments were then used to manually verify presence/absence of missing enzymes for filling pathway holes where possible. This curated data, based on both KEGG and OrthoMCL assignments, were used to generate the final mapping of enzymes to pathways, and using this info a metabolic pathway network was drawn to represent all major pathways involved in carbohydrate, energy, fatty acid, lipid, isoprene, steroid, amino acid, nucleotide, cofactor, polyamine, and redox metabolism (Figure 2-figure supplement 3).
Based on the enzymes mapped, we calculated the completeness of metabolic pathways by comparing the fraction of enzymes present for each pathway in each species. The complete set of enzymes mapped to each pathway (originally taken from KEGG and further curated to eliminate non-specific entries) is given in column B of Supplementary file 3. The fractional values were then color-coded and the resulting data are shown in Figure 2B. In order to visualize the retention, loss or gain of higher level metabolic functions, the fraction of enzymes mapped to these pathways is indicated as a pie chart for hypothetical ancestors of selected apicomplexan groups and chromerids ( Figure 2B). We used presence of enzymes across the species and the phylogenetic relationship to infer presence of enzymes in the hypothetical ancestors based on Dollo parsimony (Csuros, 2010). Dollo parsimony is based on an assumption that it is unlikely that the same enzymes were gained multiple times independently in different lineages.
Phylogeny of heme pathway enzymes, the urea pathway CPS and enzymes involved in fatty acid biosynthesis.
Predicted proteins from Vitrella (Chromera heme pathway is already published ) were searched for enzymes involved in the synthesis of tetrapyrroles (aminolevulunuic acid [ALA] synthase, ALAS; ALA dehydratase, ALAD; Porphobilinogen deaminase, PBGD; Uroporphyrinogen synthase UROS; Uroporphyrinogen decarboxylase, UROD; Coproporphyrinogen oxidase, CPOX; Protoporphyrinogen oxidase, PPOX; and Ferrochelatase FeCH). All genes identified were aligned to the homologs available in public databases such as NCBI and JGI using Muscle (Edgar, 2004), with the alignments further edited in SeaView (Gouy et al., 2010). The results from these analyses are shown in Supplementary file 4. The same procedure but searching in the predicted proteomes of both chromerids was used to construct the alignment of carbamoyl phosphate synthases (CPS). Genes coding for enzymes containing ketoacyl synthase domain were searched using BLAST. Functional domains were searched using InterProScan (Zdobnov and Apweiler, 2001).
Phylogenetic trees of all investigated enzymes were constructed using the ML approach (RAxML [Stamatakis, 2014]), Bayesian inference (PHYLOBAYES [Lartillot and Philippe, 2004]), and a method designed to deal with amino acid saturation (AsaturA [ Van de Peer et al., 2002]). ML trees were computed under the gamma corrected LG4X model of evolution as implemented in RAxML 7.4.8a using the rapid-bootstrap optimization algorithm in 1000 replicates. Bayesian phylogeny was inferred using empirical site-heterogenous model C40 as implemented in Phylobayes 3.2f. Two independent chains were run until they converged (i.e., maximum observed discrepancy was lower than 0.2), and the effective number of model parameters was at least 100 after the first 1/5 generation was omitted from topology and posterior probability inference.
AsaturA trees were computed using a Poisson corrected LG model and the support was assessed from 1000 replicates. Sequences from Vitrella (all enzymes under investigation) and Chromera (CPS and FAS enzymes) were inspected for the presence of N-terminal leader sequences using SignalP (Bendtsen et al., 2004) and TargetP (Emanuelsson et al., 2007) software respectively, suggesting targeting to either mitochondrion (with mitochondrial transit peptide) or plastid (with bipartite leader composed of ER signal peptide and transit peptide).

Fatty acid synthesis pathway
C. velia cells were grown in the f2 medium. Cultures were kept in 25 cm 2 flasks under artificial light with photoperiod 12/12, light exposure between 70 and 120 μmol/m 2 /s and temperature of 26˚C. 1 ml of C. velia stationary culture was added to each flask with 20 ml of f2 solution. The cultures were grown for one month to reach a high density of cells. Since triclosan is not soluble in water, dimethyl sulfoxid (DMSO) was used as a soluble mediator. Four experimental groups were established: control, control with DMSO, Chromera treated with triclosan in concentrations of 1 mM and 0.5 mM, respectively. After 16 days of incubation, cultures were harvested via centrifugation, and pellets were stored in −20˚C for subsequent lipid extraction. Homogenization of algal sample was achieved by Mini-beadbeater (Biospec Products).
Homogenates were dried and weighted. Lipids were extracted using on chloroform and methanol, as described before (Folch et al., 1957). An aliquot of 100 μl volume was subjected to HPLC ESI/MS. The technique was performed on an ion trap LTQ mass spectrometer coupled to Allegro ternary HPLC system equipped with Accela autosampler with the thermostat chamber (all by Thermo, San Jose, CA, USA). 5 μl of sample was injected into a Gemini column 250 × 2 mm i.d. 3 μm (Phenomenex, Torrance, CA, USA). The mobile phase consisted of (A) 5 mmol/l ammonium acetate in methanol, (B) water, and (C) 2-propanol. The analysis was completed within 80 min with a flow rate of 200 μl/min by following gradient of 92% A and 8% B in 0-5 min, then 100% A till the 12th minute, subsequently increasing the phase C to 60% till 50 min and holding for 15 min and then in at the 65th minute returning back to the 92:8% A:B mixture and 10 min to column conditioning. The column temperature was maintained at 30˚C. The mass spectrometer was operated in the positive and negative ion detection modes at +4 kV and −4 kV with capillary temperature at 220˚C. Nitrogen was employed as shielding and auxiliary gas for both polarities. Mass range of 140-1400 Da was scanned every 0.5 s to obtain the full scan ESI mass spectra of lipids. For investigation of the lipid molecules structures the collisionally induced decomposition multi-stage ion trap tandem mass spectra MS 2 in both polarity settings were simultaneously recorded with a 3 Da isolation window. Maximum ion injection time was 100 ms, and normalized collision energy was 35%. Major phospholipids, galactolipids, and neutral lipids molecular species that are detected were separated by reversed-phase HPLC. The structure of each entity was identified by MS 2 experiments in positive or negative mode. Peak areas for each detected lipid component were summarized and their relative contents estimated to sum of all obtained peaks.
Raw extracted lipids have to be transformed to methylesters of fatty acids (FAMEs) to enable application of the GC technique. For this purpose sodium methoxide was employed as a transesterification reagent, as previously described (Zahradnickova et al., 2014). FAMEs were then analyzed by GC/FID. Hydrocarbon with 26-carbon chain was chosen as an internal standard. The chromatography was performed using gas chromatograph GC-2014 (Shimadzu) equipped by with column BPX70 (SGE)-0.22 mm ID; 0.25 μm film; 30 m length. μl of derivatized sample was injected via autosampler and injector  to the column in split mode (split ratio 10). The temperature of the injector was 220˚C. The starting temperature of the column was 120˚C holding for 4 min. Then, the temperature increased to the 180˚C in at the rate of 10˚C per minute, and after that 7˚C per minute to 230˚C. Temperature of the flame ionization detector was 260˚C. The whole analysis takes took approximately 20 min. H 2 was used as a carrier gas. For the identification of particular FAs, a mixture of 37 standards purchased from Supelco Inc. was used.

Results and discussion
Global metabolic map Metabolic annotations based on ortholog assignments with KEGG and OrthoMCL database showed that chromerids contain all major primary metabolic pathways typically found in freeliving unicellular eukaryotes (Figure 2-figure supplement 3). 2918 Chromera proteins and 2985 Vitrella proteins were assigned KO numbers, from which 432 Chromera (1.3% of proteome) and 425 Vitrella proteins (1.8% of proteome), respectively, were identified as enzymes with a metabolic function based on EC number association.
In support of their autotrophic lifestyle, the chromerids appear to be capable of generating de novo all primary carbon metabolites such as the various sugars and other reduced carbon compounds (presumably via photosynthesis and associated carbon fixation pathways), amino acids, nucleotides, fatty acids and lipids, isoprene and steroid derivatives, and most vitamins (except biotin and vitamin B12). These organisms are also capable of assimilating both nitrate and sulfite and can generate energy from photosynthesis as well as mitochondrial respiration. A full complement of enzymes involved in sugar and sugar derivative metabolism, such as glycolysis, Kreb's cycle, pentose phosphate pathway, inositol mono-and poly-phosphate formation, polysaccharide formation, and amino-and nucleotide-sugar formation, is encoded by the chromerids. Chromerids are also capable of synthesizing sulfoquinovosyl-diacyl-glycerol lipids, which are found associated with the chloroplast in photosynthetic organisms. Generally, Chromera and Vitrella have similar sets of metabolic enzymes. Enzymes for the oxidative arm of pentose phosphate pathway, conversion of diacyl-glycerol to phosphatidyl ethanolamine, phosphatidyl ethanolamine to phosphatidyl serine, and XppppX to XTP are absent in Chromera, while, on the other hand, the enzymes involved in conversion of glucose-1P to UDP-glucose, and cytidine to uridine are missing in Vitrella. One major difference between the two chromerids is that the complex III of the mitochondrial respiratory chain (cytochrome c reductase) is missing in Chromera, but present in Vitrella (Flegontov et al., 2015). This feature of the Chromera mitochondrion, that is, absence of complex III but presence of complex IV, makes it unique among all mitochondria and mitochondria-derived organelles.
The crucial enzyme for the urea pathway, mitochondrially targeted carbamoyl phosphate synthase (CPS) (Allen et al., 2011), is absent from both chromerids. However, while Chromera contains single CPS involved in pyrimidine biosynthesis, Vitrella genome encodes two CPSs. But both these genes are closely related suggesting they are recent duplicates (ML tree is shown in Figure 2-figure supplement 2A) and they lack a mitochondrial leader sequence at the N-terminus (data not shown). This means that Vitrella duplicated CPSs are not likely to be involved in urea cycle. In contrast to Vitrella, Chromera lacks the gene encoding argininosuccinate lyase (ASL), an enzyme of the ornithin cycle.

Plastid-related metabolic pathways
Chromerid photosystems have a reduced set of genes similarly to that of other algae with a complex plastid. The patterns of reduction were lineage-specific, even between the two known chromerids. We found psbM, petN, psaJ, Psb27, Ycf4, and Ycf44 genes in Vitrella but absent in Chromera; vice versa, Ycf39 and Ycf54 are absent in Vitrella but present in Chromera (Supplementary file 3). This demonstrates that the plastid of Chromera is more diverse and reduced when compared to Vitrella with respect to both the composition of photosystems and the number of genes encoded in the plastid genome (Janouškovec et al., 2010). In spite of substantial reduction of photosystems, photosynthesis in Chromera is highly efficient (Quigg et al., 2012).
The heme pathway in Vitrella is homologous to that found in Chromera  for most of the involved enzymes (ALAS, ALAD, PBGD, UROS, PPOX), however, Vitrella and Chromera do not constitute sister groups in the CPOXs and FeCH trees (trees not shown). Some enzymes are not present in single copies (UROD) in Vitrella, in contrast to Chromera, where three orthologs originating from cyanobacteria, endosymbiont nucleus, and exosymbiont nucleus are present . For some investigated enzymes (UROS, UROD, CPOX), only the endoplasmic reticulum signal peptide was found with transit peptide missing from the sequence, suggesting their possible location in the endoplasmic reticulum or periplastidal space.
Genes containing the ketoacyl synthase domain and thus likely involved in fatty acid or polyketide synthesis were searched in the genomes of chromerids. We found that both algae possess multi-modular enzymes responsible for fatty acid synthesis type I (FASI), similar to some apicomplexan parasites, such as Cryptosporidium spp. and T. gondii. The longest multimodular enzyme found in Chromera contains five multi-domain modules, reaching over 11,000 amino acids in length (Figure 2-figure supplement 2B).

Evolution of metabolic pathways
Apicomplexan parasites differ drastically from each other in their metabolic functions, and have a significantly reduced metabolic capability in comparison to the chromerids. Apicomplexans are non-photosynthetic and therefore lack all associated metabolic activities including photosynthetic carbon fixation. Interestingly, however, all plastid-bearing parasites have retained only the ferredoxin-NADP+ reductase (FNR)/ferredoxin redox system of the photosynthetic electron transport (Lim and McFadden, 2010). In photosynthetic organisms, this redox system mediates the transfer of electrons originating from water to NADP+, resulting in the formation of NADPH (cofactor for fatty acid biosynthesis and Calvin cycle), and it is likely that this role is conserved in chromerids. In apicomplexans, the source of electrons for generating reduced ferredoxin is not clear, but it is evident that reduced ferredoxin is required for generating reducing equivalents and is a cofactor for several plastid-associated enzymes, including those involved in isoprene synthesis (Lim and McFadden, 2010). Other notable pathways missing in apicomplexans but present in the chromerids include the following: glyoxalate pathway; steroid biosynthesis; synthesis of aromatic and branched-chain amino acids; purine synthesis; and synthesis of cofactors such as thiamine, riboflavin, and nicotinate ribonucleotide.
Despite the reduced metabolic capabilities, certain core metabolic functions are conserved in chromerids as well as in all apicomplexan parasites, and many of these are likely to be essential. These pathways include: glycolysis; synthesis of ubiquinone, inositol-P derivatives, GPI-anchor, mono-, di-and tri-acyl glycerol, isoprene derivatives, and N-glycans; a subset of scavenge reactions for purine and pyrimidine bases and their conversion to nucleotides; glycine-serine inter-conversion; one-carbon folate cycle and S-adenosyl-methionine formation. There are many metabolic pathways that are retained in specific apicomplexan lineages but shared with the chromerids (see Figure 2B and Figure 2-figure supplement 3). The following are notable examples of pathways shared between chromerids and a apicomplexan lineage: with Plasmodium, polyamine synthesis; with Cryptosporidium, conversion of serine to tryptophan; with Toxoplasma, branched-chain amino acid degradation, synthesis of aspartate, lysine, and methionine; synthesis of molybdopterin, biopterin, pyridoxal-phosphate, and pantothenate cofactors. Surprisingly, with respect to the chromerids and other apicomplexans, Cryptosporidium appears to be the only apicomplexan lineage to have gained a metabolic function of conversion of thymidine to dTMP by thymidine kinase. In addition, the type I and II pathways for fatty acids biosynthesis show lineage-specific distribution in apicomplexans and chromerids ( Figure 2-figure supplement 3).
We can also find example of pathways that have been lost in a lineage-specific manner. For example, the ability to synthesize the di-saccharide trehalose is missing only in Plasmodium. However, the most dramatic loss of metabolic function in a single lineage can be found in cryptosporidia. These parasites are devoid of all plastid-and mitochondria-associated metabolic functions and other pathways involved in the synthesis of ribose-P, pyrimidine, most amino acids, heme, fatty acids (de novo), and isoprene units. It seems that the lack of mitochondrial oxidative pathways in cryptosporidia led to loss of the ability to generate flavin nucleotide (FMN/FAD) and lipoic acid cofactors.
In order to cope with loss of metabolic pathways, parasites have evolved various mechanisms for scavenging the required nutrients and metabolites from their respective hosts. For example, metabolites such as heme, fatty acids, steroids (specifically cholesterol), and sphingolipids are known to be scavenged by various apicomplexans as needed from their respective hosts.
According to the metabolic pathway maps, certain metabolic functions, which are coupled to each other, have been either retained or lost concomitantly in various species (Figure 2-figure supplement 3). For example, piroplasms and cryptosporidia lack de novo fatty acid biosynthesis along with the pyruvate dehydrogenase enzyme complex (plastidassociated in apicomplexans), which is known to supply acetyl CoA units for fatty acid synthesis. On the other hand, these parasites have retained the ability to convert pantothenate to co-enzyme A, which is required for the activation of fatty acids scavenged from the hosts (Leonardi et al., 2005). Similarly, activities of the serine hydroxyl methyl transferase and thymidylate synthase enzymes are coupled to each other and to one-carbon folate metabolism. Therefore, these three metabolic functions are retained in all parasites.
idea, some cases of reduction were not limited to Apicomplexa, and could be observed in the sister phyla of the ciliates and dinoflagellates.
This pattern of loss raises the question of what losses correlate with the transition to parasitism and which are pre-adaptive, arising more deeply in the lineage. The unique phylogenetic position of chromerids (Janouškovec et al., 2010(Janouškovec et al., , 2012Oborník et al., 2012) allows finer dissection of the patterns of retention/loss observed previously. Hence, we chose to focus on detailed characterization of the three previously studied sets of membrane trafficking machinery in the predicted proteomes of Chromera and Vitrella, together with 24 closely related organisms for comparison.

ESCRT machinery
The ESCRT machinery is a set of five sub-complexes involved in recognition of ubiquitylated proteins and recruitment to the multi-vesicular body (MVB)/late endosome for degradation (Leung et al., 2008). Most eukaryotes, including Chlamydomonas reinhardtii and our representative stramenopile taxa (Thalassiosira pseudonanna, Phaeodactylum tricornutum, Ectocarpus siliculosus, and Pythium ultimum), have a complete set of the ESCRT machinery, suggesting that the ancestor of alveolates, and indeed the Last Eukaryotic Common Ancestor (LECA), likely had it. Though this ancestral complement appears to have been reduced in ciliates in the ESCRTI and III complexes, and a few components are missing from dinoflagellate taxa, numerous gene duplications have occurred as well, suggesting sculpting of the machinery. By comparison, apicomplexan parasites exhibit significant reductions in their ESCRT machinery (Leung et al., 2008). Cryptosporidia, coccidia, and plasmodia appear to lack any subunits of the ESCRTI and II complexes. ESCRTIII conservation is better, though no apicomplexan encodes Vps24, and multiple taxa have lost Vps20 as well. A similar pattern is seen for the ESCRTIII-a machinery, with piroplasmids encoding only Vps46 and Vps4. Coccidia additionally encode Vps31, and cryptosporidia Vps60, whereas plasmodia encode all subunits (rodent parasites like Plasmodium chabaudi), or lack Vps31 (human or simian parasites like P. falciparum). Chromera and Vitrella possess all ESCRT subunits except for the ESCRT-III component CHMP7, which is rarely found outside the opisthokont supergroup (Leung et al., 2008). This observation suggests two conclusions regarding the evolution of the ESCRT machinery within alveolates: massive gene loss within the Apicomplexa occurred recently, after the split from the proto-apicomplexan ancestor, and some losses of machinery shared between apicomplexans and other alveolates are due to independent losses. An excellent example of this latter case is that of Vps37, which is present only in chromerids, but in no other alveolate included in the current study, suggesting its function was dispensable in a large number of lineages.

APs
The APs are heterotetrameric complexes that select cargo for inclusion into transport vesicles at organelles of the late secretory system and endocytic system. AP1 and AP3 are involved in the transport between the trans-Golgi network (TGN) and endosomes. AP2 is involved in the transport from the cell surface. AP4 is involved in TGN transport to either endosomes or the cell surface, while the recently described AP5 complex is involved in the transport from late endosomes back to early endosomes. All five complexes are ancient, having likely been present in the LECA (Nevin and Dacks, 2009;Hirst et al., 2011). However, the complexes have also been secondarily lost on multiple occasions as well. Outgroup taxa in our data set possess AP1-4 complexes, with the exception of C. reinhardtii lacking AP3, but only Symbiodinium minutum possesses an AP5 complex.
Apicomplexa display higher variability in AP complex retention. With the exception of AP2M in cryptosporidia, all taxa retain full AP1, 2, and 4 complexes. Piroplasms lack all subunits of the AP3 complex, and together with P. falciparum and Plasmodium reichenowi, lack AP5 as well.
Other plasmodia possess all AP5 subunits with the exception of the mu subunit. This result was unexpected, based on the usual patterns of conservation seen across Plasmodium species.
Presence of AP5 in the majority of these organisms suggests the exciting possibility of a novel trafficking pathway absent from the comparatively well-studied human parasite P. falciparum. Additionally, our increased taxon sampling has suggested that AP5 may be well conserved across Myzozoa, a result otherwise indeterminable from previous studies of this protein family (Hirst et al., 2011). Cryptosporidia also lack AP3, but unlike piroplasmids, they possess almost a complete AP5 complex, missing only the sigma subunit. Coccidia are the exception, possessing all five AP complexes in their entirety. Excitingly, Chromera and Vitrella, like coccidia, possess a complete complement of adaptin subunits, suggestive of a more complete set of trafficking pathways to endosomal organelles in these organisms.

MTCs
The MTCs are an assembly of heteromeric protein complexes involved in the first stage of vesicle fusion and delivery of contents from a transport vesicle to a destination organelle. Each one is specific to an organelle or transport pathway and all eight complexes have been deduced as present in the LECA, with some interesting cases of secondary loss. While C. reinhardtii and the stramenopiles encode a complete set of MTC machinery, several of these MTCs have interesting patterns of conservation, specifically in the Apicomplexa (Klinger et al., 2013a).
The conservation of the TRAPP I-II complexes is unclear through eukaryotes and clear patterns are difficult to draw. However, the apparent absence of the entire TRAPPII complex in Vitrella may be due to gaps/biases/absences in sequencing, protein prediction, or analysis, but has interesting ramifications if proven to be a real biological phenomenon.
Exocyst is involved in diverse processes, all of which involve polarized exocytosis (Liu and Guo, 2012). Tetrahymena appears to encode only four of the Exocyst subunits. None of the eight subunits were identifiable in Chromera, Vitrella, nor in any of the Apicomplexa or dinoflagellates. This confirms, and extends, a previous result suggesting the absence of this complex within the Myzozoa, suggesting a bona fide ancestral loss concurrent with the acquisition of an apical complex that could have served an analogous tethering function for secretory organelles.
COG is an octameric complex involved in tethering at the Golgi body (Tomavo et al., 2013;Klinger et al., 2013b). The COG complex is poorly conserved in Apicomplexa and a ciliate Tetrahymena thermophila only encodes half of the COG subunits. In contrast, all eight COG subunits are present in Chromera and Vitrella. The retention of a complete COG complex in both Chromera and Vitrella contrasts with the substantial loss of subunits in Apicomplexa, especially outside the coccidians (Klinger et al., 2013b) (Figure 2-figure supplement 4).
Notably, this conservation is consistent with the presence of robust, stacked Golgi bodies in Chromera  and T. gondii (Pelletier et al., 2002), compared to aberrant morphology in other Apicomplexa.
The complexes of CORVET and HOPS mediate tethering at the early and late endosomes (Tomavo et al., 2013;Klinger et al., 2013b). They share a core of four subunits with complexspecific proteins (Vps3 and 8 for CORVET and Vps39/41 for HOPS). Though all taxa encode the complete VpsC core of the HOPS/CORVET complex, all taxa except for T. gondii only appear to encode the CORVET-specific interactor Vps3. Chromera and Vitrella, like Apicomplexa, possess the entire VpsC core complements as well as the HOPS component Vps41 and both CORVET components.
Chromerids exhibit complex life cycles, from immotile vegetative cells to multi-cellular sporangia, and occasionally motile flagellated cells. Both lineages contain numerous potential locales for intracellular trafficking including mitochondria, plastid, starch granules, flagella, micronemes, and, in Chromera, the chromerosome. Additionally, vesicular traffic to the sporangial/cyst wall has been visualized in both lineages (Oborník et al., 2012). Our results indicate that chromerids possess an appropriately complex complement of membrane trafficking machinery to achieve these requirements.
Though MVBs have not been explicitly imaged or characterized in either lineage to date, both Chromera and Vitrella encode a complete set of ESCRT machinery, suggestive of the presence of functional MVBs. These may play a key role in modulating surface protein expression in various life cycle stages. Importantly, the close evolutionary position of Chromera and Vitrella to Apicomplexa suggests that the extensive decrease in ESCRT subunit conservation in Apicomplexa occurred in the immediate ancestor and is not an ancestral feature of a more inclusive group (Leung et al., 2008) (Figure 2-figure supplement 4). Particularly, the lack of some ESCRT subunits such as Vps37 in ciliates and dinoflagellates is most parsimoniously attributed to multiple independent losses. Further evidence for a complete set of ESCRT machinery in the last common alveolate ancestor comes from the conservation of all subunits to the exclusion of CHMP7 in the outgroup stramenopile taxa and in C. reinhardtii. The absence of CHMP7 in all taxa is not unusual, as it is lost in numerous taxa across eukaryotes (Leung et al., 2008).
Conservation of adaptin subunits is striking, particularly the complete retention of AP5 in chromerids. In an initial study of seven organisms from the SAR supergroup (the group in which chromerids belong to), only two (B. natans and T. gondii) were found to encode the complex; conservation across eukaryotes was similarly sparse (Hirst et al., 2011). The presence of a complete AP5 complex in chromerids and coccidians may be indicative of a conserved function in both lineages. Likewise, the retention of an almost complete AP5 in cryptosporidia and plasmodia may have functional significance or may simply represent a reductive evolutionary process that has not yet reached completion. The complete lack of AP5 in P. falciparum and P. reichenowi supports the latter view. As with the ESCRT complexes, the presence of AP1-5 in chromerids suggests the loss of AP3 and AP5 observed in some Apicomplexa is secondary, as well as the loss of AP5 in Perkinsus marinus, and in both ciliate lineages.
Presence of a complete VpsC core along with an additional CORVET subunit Vps3 in the majority of apicomplexan genomes suggests the potential for a modified HOPS/CORVET complex that interacts with Rab5 to direct tethering at the micronemes/rhoptries. This is in keeping with the view of rhoptries/micronemes as divergent endolysosomal organelles (Klinger et al., 2013b). However, chromerids do not appear to possess rhoptries, although chromerids possess cellular components analogous to micronemes (Oborník et al., , 2012. More HOPS/CORVET subunits were found to be conserved in T. gondii, which are the only apicomplexan to date to be described as possessing a canonical lysosome-like compartment 5 , suggesting that complete complexes are retained in these lineages because they are required for trafficking to canonical lysosome-related organelles as well. Additionally, Chromera possesses the chromerosome, which often displays intralumenal vesicles similar to MVBs, suggesting it may also be derived from endosome-like organelles . In conclusion, apicomplexans possess unusual endomembrane compartments including atypical Golgi and endosome-derived invasion organelles such as micronemes and rhoptries (Klinger et al., 2013b). Modifications in the complement of membrane trafficking machinery, including the loss of key protein complexes found in most eukaryotes, have been observed in the apicomplexan lineage, potentially associated with the specialization of the endomembrane system. The absence of some components (Exocyst, Vps39, Trs120, Tip20) within Chromera and Vitrella suggests pre-adaptation to parasitism deeper in the apicomplexan lineage. By contrast, the presence of near complete complements of key machinery (AP1-5, ESCRTs, COG) absent in many apicomplexans, pinpoints the timing of the losses at the colpodellid/apicomplexan transition.
apicomplexans and lack of Arp2/3 complex suggest their importance, perhaps reflecting a switch from Arp2/3 complex to formins for actin nucleation during the evolution of Apicomplexa. Taken together, it seems that an Arp2/3 independent actin nucleation mechanism had already evolved before Apicomplexa and chromerids, and losses of ARP have probably begun too, although inferring the exact timing and sequence of losses will require studying more closely related species such as Colpodella.
We analyzed coronins, a major conserved gene family with a multifunctional role in actin regulation and vesicular transport (Rybakin and Clemen, 2005). These are WD40-repeat containing proteins, which represent the only candidate for actin bundling in apicomplexan parasites. Coronins inhibit the nucleating activity of Arp2/3, unlike other known Arp2/3-binding proteins. We observed absence of coronins in both chromerids, which is consistent with the notion that they, functionally linked to the Arp2/3 complex, were lost ( Figure 2-figure  supplement 5C). Although parasite homologs do not have the microtubule-binding domain of canonical coronins, but essential amino acid residues are conserved (Gandhi et al., 2010). Thus, coronin could be playing a role in stabilizing F-actin scaffolds or having an alternative role in vesicular transport in apicomplexan parasites.
Profilins are actin-binding proteins that supply pools of readily polymerizable actin monomers . Genes encoding profilins were found in all 26 species studied except for an oomycete (P. ultimum) and diatoms (P. tricornutum, Thalassiosira pseudonana). Apicomplexaspecific profilins have βmini1 and βmini2 domains, which provide an extended interface with actin and formed the structural basis of their actin-binding function in Toxoplasma (Kucera et al., 2010) and Plasmodium (Kursula et al., 2008). These domains are not found in other eukaryotes. Sequence alignment of these profilins reveals an intriguing observation that Vitrella (Vbra_7301.t1) had these β-domains previously thought to be specific to Apicomplexa, with partial conservation in Chromera (Cvel_18957.t1) and in dinoflagellate P. marinus (XP_002774080). The β-domains were not detected in other non-apicomplexan species. All species studied has had only one profilin gene except for chromerids where we observed 2 in Chromera and 3 in Vitrella, including an one-to-one ortholog of the apicomplexan profilin in both chromerids. The F-actin capping, CapZ duplex, a dimer of αand β-CPs, prevents polymerization from the 'barbed' (plus) end. It is conserved across Apicomplexa except for in piroplasms. It is also conserved in most of the species studied including stramenopiles, dinoflagellates, and both chromerids. In Apicomplexa, several gelsolin domain-containing proteins were found but they are unlikely to be functionally related and are speculated to be Sec23/Sec24-like proteins, which function in vesicular transport .
Cofilin/ADF genes promote de-branching of actin filaments and are well conserved among species studied. However, plasmodia differ from the rest of the Apicomplexa species in having an additional copy of the ADF gene. Phylogenetic analysis shows that ADF in plasmodia has duplicated and diverged with respect to the rest of the Apicomplexa, and recent structural studies explain the mechanism of action of Plasmodium ADFs (Singh et al., 2011;Wong et al., 2014). This represents a clear example of additional innovations of actin regulation in certain apicomplexan clades.
In addition, we identified myosin families in the 26 species using a myosin HMM model (Foth et al., 2006) (Figure 2-figure supplement 5C). Members of piroplasmids such as Theileria annulata and Theileria parva have the fewest genes among the apicomplexan species examined, likely because piroplasms do not require motility for intracellular invasion. On the other hand, we detected the most complete myosin family repertoire in Chromera and Vitrella. We detected certain myosin families in some apicomplexan genera, but not among nonapicomplexan species, indicating lineage-specific gains (data not shown). In summary, combinations of lineage-specific losses and gains have led to streamlined, unique repertoires of actins and myosins in various apicomplexan species.