MBE Advance Access originally published online on May 23, 2007
Molecular Biology and Evolution 2007 24(8):1832-1842; doi:10.1093/molbev/msm101
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Research Articles |
Plastid Genome Sequence of the Cryptophyte Alga Rhodomonas salina CCMP1319: Lateral Transfer of Putative DNA Replication Machinery and a Test of Chromist Plastid Phylogeny
* Genome Atlantic and the Canadian Institute for Advanced Research, Program in Evolutionary Biology, Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada
The Atlantic Genome Centre, Halifax, Nova Scotia, Canada
Email: jmarchib{at}dal.ca.
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Abstract |
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 TOP Abstract IntroductionMaterials and MethodsResults and DiscussionConclusionSuppementary MaterialAcknowledgementsReferences |
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Cryptophytes are a group of unicellular algae with chlorophyll c–containing plastids derived from the uptake of a secondary (i.e., eukaryotic) endosymbiont. Biochemical and molecular data indicate that cryptophyte plastids are derived from red algae, yet the question of whether or not cryptophytes acquired their red algal plastids independent of those in heterokont, haptophyte, and dinoflagellate algae is of long-standing debate. To better understand the origin and evolution of the cryptophyte plastid, we have sequenced the plastid genome of Rhodomonas salina CCMP1319: at 135,854 bp, it is the largest secondary plastid genome characterized thus far. It also possesses interesting features not seen in the distantly related cryptophyte Guillardia theta or in other red secondary plastids, including pseudogenes, introns, and a bacterial-derived gene for the tau/gamma subunit of DNA polymerase III (dnaX), the first time putative DNA replication machinery has been found encoded in any plastid genome. Phylogenetic analyses indicate that dnaX was acquired by lateral gene transfer (LGT) in an ancestor of Rhodomonas, most likely from a firmicute bacterium. A phylogenomic survey revealed no additional cases of LGT, beyond a noncyanobacterial type rpl36 gene similar to that recently characterized in other cryptophytes and haptophytes. Rigorous concatenated analysis of 45 proteins encoded in 15 complete plastid genomes produced trees in which the heterokont, haptophyte, and cryptophyte (i.e., chromist) plastids were monophyletic, and heterokonts and haptophytes were each other's closest relatives. However, statistical support for chromist monophyly disappears when amino acids are recoded according to their chemical properties in order to minimize the impact of composition bias, and a significant fraction of the concatenate appears consistent with a sister-group relationship between cryptophyte and haptophyte plastids.
Key Words: cryptophytes • plastid • chromists • chromalveolates • secondary endosymbiosis
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionSuppementary MaterialAcknowledgementsReferences |
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Plastids (chloroplasts) evolved from free-living cyanobacteria within the confines of a nonphotosynthetic host eukaryote (Gray and Doolittle 1982
). The "primary" endosymbiotic origin of plastids is widely believed to have occurred only once (Moreira, Le Guyader, and Phillippe 2000; Palmer 2003; Keeling 2004; Rodriguez-Ezpeleta et al. 2005) and 3 modern-day eukaryotic lineages—red algae, green algae (including land plants), and glaucophytes—harbor plastids stemming directly from this landmark event (Palmer 2003; Keeling 2004). More recently, the plastids of red and green algae have spread laterally across the eukaryotic tree via "secondary endosymbiosis," i.e., the uptake of a photosynthetic eukaryote by an unrelated nonphotosynthetic host (Delwiche 1999; Archibald and Keeling 2002, 2005; Palmer 2003; Keeling 2004). Unlike primary plastids, which are surrounded by 2 membranes, secondary plastids are characterized by the presence of 3 or 4 bounding membranes, a feature that complicates the import of nucleus-encoded, plastid-targeted proteins in secondary plastid-containing organisms (Cavalier-Smith 1999; McFadden 1999; Soll and Schleiff 2004). Some of the most abundant and ecologically significant eukaryotic phototrophs on Earth acquired their plastids secondarily, yet many of the details surrounding the pattern and process of secondary endosymbiosis remain unclear.
Determining the number of endosymbioses that gave rise to the known spectrum of red algal–derived plastids has been particularly challenging, in large part due to the tremendous morphological, biochemical, and molecular diversity exhibited by the organisms bearing them. These include heterokonts (e.g., diatoms and kelp), haptophytes, dinoflagellates, and (probably) apicomplexan parasites, the latter group containing a highly derived nonphotosynthetic organelle whose precise origin has been difficult to discern (reviewed by Delwiche 1999; Archibald and Keeling 2002, 2005; Palmer 2003; Keeling 2004). The cryptophytes are a ubiquitous group of flagellated unicells that also possess secondary plastids of red algal origin. Together with the chlorarachniophytes, an unrelated group of algae with green algal secondary plastids (Ishida, Green, and Cavalier-Smith 1999; Keeling 2004; Gilson et al. 2006; Rogers et al. 2007), cryptophytes are unlike other secondary plastid–containing algae in that the primary endosymbiont nucleus—the "nucleomorph"—persists in the remnant cytosol of the engulfed algal cell between the inner and outer pairs of plastid membranes (Gilson, Maier, and McFadden 1997; Archibald 2007). Nucleomorph (Douglas et al. 2001) and plastid (Douglas and Penny 1999) genome sequence data from the model cryptophyte Guillardia theta convincingly show a red algal ancestry for the cryptophyte plastid, although the precise relationship between cryptophyte plastids and other red secondary plastids is not known.
Based on the unique membrane topology of their plastids and the shared presence of the photosynthetic pigment chlorophyll c2, Cavalier-Smith (1982, 1986) placed the cryptophytes, heterokonts, and haptophytes together in the kingdom Chromista, hypothesizing that photosynthesis evolved in these organisms only once as a result of a single endosymbiosis in their common ancestor. More specifically, cryptophytes have been argued to be the deepest branching of the 3 groups, with the haptophytes and heterokonts having diverged from one another more recently (Cavalier-Smith 2003). The so-called "chromalveolate" hypothesis goes 1 step further in postulating that the endosymbiosis that gave rise to the chromist plastid occurred even earlier, in a common ancestor these organisms shared with dinoflagellates and apicomplexans (Cavalier-Smith 1999, 2004). The main rationale behind this idea is to invoke the fewest number of secondary endosymbioses possible, given that each plastid acquisition requires extensive nucleus-to-nucleus gene transfers and the evolution of a protein targeting system (Cavalier-Smith 1999; McFadden 1999). However, inferring the minimum number of secondary endosymbioses has important implications for how we interpret the evolutionary history of a significant fraction of eukaryotic biodiversity. This is because many dinoflagellates and heterokonts are aplastidic and/or nonphotosynthetic, and the ciliates, which together with dinoflagellates and apicomplexans comprise the alveolates, are an entirely nonphotosynthetic lineage. If correct, the chromalveolate hypothesis demands that all dinoflagellates, heterokonts, and ciliates evolved from plastid-bearing ancestors (for discussion see Bachvaroff, Sanchez Puerta, and Delwiche 2005; Bodyl 2005). The recently sequenced macronuclear genome of the ciliate Tetrahymena thermophila provided no evidence for a photosynthetic ancestry in ciliates (Eisen et al. 2006), although plastid-associated genes were found in the genome of the aplastidic heterokont pathogen Phytophthora (Tyler et al. 2006), suggesting that outright organelle loss is possible.
Early molecular data brought to bear on the origin(s) of chromist plastids failed to provide support for their common ancestry. Phylogenies of RuBisCO and plastid small subunit ribosomal DNA (SSU rDNA) placed cryptophytes, heterokonts, and haptophytes as independent lineages within red algae (e.g., Medlin et al. 1995; Daugbjerg and Andersen 1997; Oliveira and Bhattacharya 2000) and were interpreted as evidence that their plastids were acquired independent of one another. Early nuclear SSU rDNA phylogenies failed to unite the host components of these lineages (Bhattacharya, Helmchen, and Melkonian 1995), consistent with separate plastid origins. More recent analyses using larger concatenated datasets have provided evidence both for (Yoon et al. 2002; Rogers et al. 2007) and against (Martin et al. 2002) the hypothesis that chromist plastids are monophyletic. Analysis of endosymbiotic replacements involving nucleus-encoded, plastid-targeted proteins have provided strong evidence in favor of chromist and chromalveolate monophyly (Fast et al. 2001; Harper and Keeling 2003; Patron, Rogers, and Keeling 2004), although the significance of such replacements has been questioned (Bodyl 2005).
A consistent trend in molecular phylogenies that do recover chromist monophyly is the basal position of cryptophytes with respect to the other chlorophyll c-containing lineages (Yoon et al. 2002; Bachvaroff, Sanchez Puerta, and Delwiche 2005; Rogers et al. 2007). In contrast, a recent comprehensive survey of plastid genomes revealed an extremely rare lateral gene transfer (LGT) involving a noncyanobacterial rpl36 gene in the plastid genome of cryptophytes and haptophytes but not heterokonts, suggesting that the former 2 lineages could be each other's closest relatives (Rice and Palmer 2006). On balance, the evolutionary history of chromist plastids is controversial, and attempts to resolve the issue have been hampered by the limited amount of genome sequence data available from diverse members of the 3 groups. Combined with the considerable evolutionary distance between chromist plastids and those of red algae, such limited taxon sampling has made phylogenetic inferences especially prone to artifact.
Here we present the completely sequenced plastid genome of the cryptophyte Rhodomonas salina CCMP1319 and analyze it in the context of 15 other genomes, including 5 other chromist plastids and 4 red algae. The R. salina genome is unique in containing a laterally transferred dnaX gene encoding the tau/gamma subunit of bacterial DNA polymerase III and, unlike previously sequenced red secondary plastids, contains pseudogenes and introns. Concatenated phylogenies inferred from a dataset of 45 plastid proteins and subsets thereof provide insight into the complex nature of the phylogenetic signal in support of a common ancestry of haptophyte and heterokont plastids to the apparent exclusion of cryptophytes.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionSuppementary MaterialAcknowledgementsReferences |
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Genome Sequencing, Assembly, and Annotation
Six fosmid library clones with inserts derived from the R. salina plastid genome were identified in a previous study (Khan et al. 2007
). These were subcloned and sequenced to 
8x coverage using ET terminator chemistry and MegaBace capillary DNA sequencers as described therein. Based on synteny shared with the plastid genome of G. theta (Douglas and Penny 1999), exact match and degenerate primers were designed to genomic regions between psaB and rpl13, and long-range PCR was used to amplify the remaining regions of the R. salina genome. High-fidelity Taq polymerase was used, and PCR products were purified using the MinElute Gel Extraction Kit (Qiagen Sciences, Valencia, CA). PCR products were cloned using the Topo TA Cloning Kit (Invitrogen) according to the manufacturer's protocol, and at least 3 independent clones were sequenced per amplicon. Plasmids were isolated using the Fastplasmid Mini Kit (Eppendorf AG, Hamburg, Germany) and sequenced using the CEQ Dye Terminator Cycle Sequencing (DCTS) kit (Beckman Coulter, Inc., Fullerton, CA) and run on Beckman Coulter CEQ8000 capillary DNA sequencers. Sequences were assembled into contigs using Staden (Staden 1996). Protein genes were annotated using NCBI ORF-finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) and BLASTX and BLASTN searches at NCBI (http://www.ncbi.nlm.nih.gov/). Ribosomal RNA genes were annotated via comparison to previously published rRNA sequences, and tRNA genes were identified using tRNAScan (http://www.genetics.wustl.edu/eddy/tRNAscan-SE/). The circular genome map was constructed using CIRDNA (http://bioweb.pasteur.fr/seqanal/interfaces/cirdna.html). The complete R. salina genome is available from GenBank (EF508371
[GenBank]
).
dnaX Amplification, Cloning, and Sequencing
Cryptophyte cell cultures were obtained from the Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP) and the Roscoff Culture Collection (RCC) and grown under conditions described previously (Khan et al. 2007). Based on the R. salina dnaX sequence and the groEL and psb28 genes flanking it, degenerate PCR primers were designed to amplify dnaX-coding regions from additional Rhodomonas species (R. baltica RCC350, R. salina CCMP1170, and Rhodomonas sp. CCMP1178). PCR products were cloned and sequenced as described above.
Phylogenetic Analyses
A concatenated dataset of 45 plastid-encoded protein sequences common among 15 algae and 2 cyanobacteria (Synechocystis sp. PCC6803 and Prochlorococcus marinus subsp. pastoris str. CCMP1986) were aligned using ClustalX (Chenna et al. 2003) and MacClade 4.06 (Maddison and Maddison 2003). The 45 genes included in the analysis were atpA, atpB, atpE, atpF, atpH, petA, petB, petD, petG, psaA, psaB, psaC, psaJ, psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbN, psbT, rpl2, rpl14, rpl16, rpl20, rpoA, rpoB, rps2, rps3, rps4, rps7, rps8, rps11, rps12, rps14, rps18, rps19, ycf3, ycf4, and psbZ. PhyML 2.4 (Guindon and Gascuel 2003) and IQPNNI (Vinh le and Von Haeseler 2004) were used to perform ML analysis on individual proteins and on the concatenated set of 9,081 amino acid positions using the cpREV, WAG, and JTT amino acid substitution matrices with a gamma distribution approximated by 4 or 8 categories to model site rate heterogeneity. Statistical support for individual branches was examined by bootstrapping (100 replicates). To reduce systematic errors associated with saturation and homoplasy, the fastest-evolving sites were determined using Tree-Puzzle version 5.2 (Strimmer and von Haeseler 1996). Trees were constructed from the concatenated alignment with sites corresponding to rates 6, 7, and 8 removed, recognizing that site rate assignments can vary slightly depending on the tree topology considered. Bayesian analyses were performed using PhyloBayes with the site-heterogeneous CAT model as described in Lartillot, Brinkmann, and Philippe (2007) using 4 gamma-distributed rate categories. Invariant sites were removed prior to analysis. We also used Tree-Puzzle to perform a site-by-site likelihood analysis of all 9,081 amino acid positions in the full dataset given 2 tree topologies, one in which cryptophytes and haptophytes were each other's closest relatives, the other where haptophytes and heterokonts shared a common branch. Differences in log likelihood were plotted in order to assess support for the 2 trees. These and other trees were compared to one another using the site likelihoods calculated above and "approximately unbiased" (AU) tests of significance. AU tests were performed using CONSEL 0.1 (Shimodaira and Hasegawa 2001) with default scaling and replicate values. Protein sequences were "recoded" according to Hrdy et al. (2004). Hydrophobic (MILV) and aromatic (FYW) amino acids were combined into a single category, and cysteine was coded as "missing data." This allowed use of PAUP (Swofford 2002) to perform ML analyses on the recoded alignment using the general-time-reversible (GTR) matrix with 4 rate categories. The frequency of recoded amino acids, as well as the proportion of invariable sites parameter was estimated from the data.
The R. salina plastid-encoded dnaX protein was used as a query to identify and retrieve a diverse set of dnaX and replication factor C proteins from public databases. Sequences were aligned using MacClade 4.06 (Maddison and Maddison 2003), manually adjusted, and analyzed using PhyML and IQPNNI as described above. The dnaX-only alignment contained 72 sequences and 240 unambiguously aligned residues. AU tests were performed as described above.
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Results and Discussion |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionSuppementary MaterialAcknowledgementsReferences |
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Rhodomonas salina Plastid Genome Sequence
The complete R. salina plastid genome is 135,854 bp (fig. 1), the largest secondary plastid genome characterized thus far. The overall G+C content is
34%, and like other chromist and red algal plastid genomes (Kowallik et al. 1995; Douglas and Penny 1999; Ohta et al. 2003; Puerta, Bachvaroff, and Delwiche 2005), the R. salina genome possesses highly similar inverted rDNA cistrons with a G+C content of 50%. The R. salina genome is predicted to encode 183 genes, including rRNAs and 31 tRNA genes. The tRNA (M) CAU (not present in the cryptophyte G. theta) is found in R. salina, and tRNA (S) GGA is substituted with tRNA (S) GCU in G. theta. The ochre termination codon TAA is used in R. salina 79% of the time, with amber (TAG) and opal (TGA) codons being used 18% and 3%, respectively. In 9 cases a valine start codon (GTG) is used rather than methionine (chll, rbcS, rpl23, rpl24, rps8, rps13, ycf27, ORF99, and dnaB). Five instances of overlapping genes are found in R. salina, many of which are also found in other chromist plastid genomes. The psbD-psbC overlap found in R. salina exists in all sequenced chromist genomes, although the amount of overlap varies. Overlaps involving atpD-atpF and rpl4-rpl23 are common to heterokonts and cryptophytes, but not haptophytes. Single nucleotide overlaps between rpl16-rpl29 and orf142-orf146 are present in R. salina, the former being found only in G. theta.
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Two very short regions of the R. salina genome could not be sequenced despite repeated attempts using varied sequencing parameters and progressively smaller subcloned fragments as template. These areas presumably correspond to regions forming extensive secondary structure. Using restriction enzyme digestion, the sizes of the gaps were determined to be
10 bp (between rps10-petF) and 100 bp (between ftsH25-ycf33) in size. Given their small size and the fact that all the genes present in G. theta (except for hypothetical ORFs) are accounted for in R. salina, it is highly unlikely that they harbor additional genes.
Overall, the R. salina plastid genome shows a high degree of synteny with the previously sequenced genome of the cryptophyte G. theta (Douglas and Penny 1999). There are 12 genes present in R. salina that are absent in G. theta, and 8 genes (mostly hypothetical ORFs) are present in G. theta and not in R. salina (supplementary table 1). HlpA, a histone-like protein previously found only in the G. theta plastid genome, is also in R. salina and the red algae Cyanidioschyzon merolae (Ohta et al. 2003) and Galdieria sulphuraria, but absent in haptophytes and heterokonts and nucleus-encoded in apicomplexans (Hall et al. 2002; Nierman et al. 2005; Pain et al. 2005). Additional genes present in cryptophytes but absent in heterokonts and/or haptophytes include cpeB, ilvB, ilvH, and infB (supplementary table 1). Almost all of the genes encoded in the genome of the haptophyte Emiliania huxleyi (Puerta, Bachvaroff, and Delwiche 2005) are present in the R. salina and G. theta genomes, while several genes present in heterokont plastids are not found in haptophytes and cryptophytes.
An intriguing feature of the R. salina plastid genome is the presence of 2 group II introns, none of which are present in G. theta (Douglas and Penny 1999) or any other chromist plastids (Oudot-Le Secq et al. 2007). A previous study identified a putative "twintron" in the groEL gene in another Rhodomonas species (Maier et al. 1995), although the R. salina groEL gene presented here lacks an intron. One of the R. salina introns resides within the photosystem gene psbN, while the second intron, located between ycf37 and ycf12 (fig. 1), appears to be degenerate (the evolution of the R. salina introns will be presented in detail elsewhere). Surprisingly, the R. salina plastid genome encodes remnants of 3 subunits of light-independent protocholorophyllide reductase (LIPOR; 
chlL, chlN, chlB; fig. 1), an enzyme involved in the light-independent synthesis of chlorophyll (Shi and Shi 2006). These genes are not found in the G. theta plastid genome nor in any secondary plastids of red algal origin, but are present in the red alga Porphyra purpurea (Reith and Munholland 1995), cyanobacteria (Kaneko et al. 1996), glaucophytes (Stirewalt 1995), and numerous organisms belonging to the green algal lineage, including land plants (Martin et al. 1998, 2002) (supplementary table 1). A comparison of the salient features of the R. salina plastid genome compared to those of other chromists and red algae is presented in supplementary table 1.
Lateral Gene Transfer
The most unexpected finding in the R. salina plastid genome is the presence of a gene with strong similarity to dnaX, which encodes the tau/gamma components of bacterial DNA polymerase (Blinkova et al. 1993; Dallmann and McHenry 1995). Although plastid DNA polymerases have been purified and characterized enzymatically (e.g., Gaikwad, Hop, and Mukherjee 2002), the process of plastid DNA replication is very poorly understood, and the enzymes directly involved are invariably nucleus-encoded. The R. salina gene represents the first instance of a putative DNA polymerase enzyme encoded in plastid DNA. Using degenerate PCR primers designed to the R. salina dnaX and to 2 genes flanking it (groEL and psb28; fig. 1), we successfully amplified dnaX from several additional Rhodomonas plastid genomes, including species CCMP1178, one of the deepest branching lineages in the Rhodomonas cluster (Lane et al. 2006).
Phylogenetic analysis reveals that the Rhodomonas dnaX genes were acquired by LGT. Analysis of dnaX proteins together with their closest eukaryotic homologs, i.e., components of replication factor C (Waga and Stillman 1998), clearly indicates that Rhodomonas dnaX is derived from the former and not the latter (fig. 2A). A more focused analysis of a larger set of dnaX homologs in isolation (fig. 2B) failed to definitively identify the donor of the gene, although in most of our analyses the Rhodomonas sequences branch (with weak to moderate support) with a subset of firmicutes, i.e., the parasitic mycoplasmas and related organisms. Phylogenies of the genes flanking dnaX clearly place R. salina within the chromist/red algal clade (data not shown), indicating that the LGT solely involved dnaX. Significantly, Rhodomonas dnaX appears distantly related to homologs in cyanobacteria, eliminating the (remote) possibility that the gene is an ancestral plastid gene. It is nevertheless intriguing that with most phylogenetic methods the cyanobacterial dnaX homologs branch modestly with the only other eukaryotic sequences in the dnaX cluster, the so-called "STICHEL" proteins of Oryza sativa and Arabidopsis thaliana. These nucleus-encoded, apparently land plant-specific proteins are 800 amino acids larger than dnaX and are not believed to be involved in DNA replication, but are instead thought to play a role in plant cell morphogenesis (Ilgenfritz et al. 2003). Putative nuclear localization signals suggest that STICHEL is targeted to the nucleus, and not plastid-localized (Ilgenfritz et al. 2003). AU tests of significance further support the idea that Rhodomonas dnaX is not specifically related to cyanobacterial dnaX or to plant STICHEL proteins; such tests reject alternate trees in which the Rhodomonas sequences are placed sister to cyanobacteria (P < 0.05) and STICHEL (P < 0.05), as well as next to the Chlorobiales/Bacteroidetes clade (P < 0.01). Interestingly, a variety of other positions for Rhodomonas dnaX within the noncyanobacterial/plant portion of the tree (e.g., alternate placements within and between Firmicutes and Proteobacteria) are not significantly worse than the tree shown in figure 2B. It is thus possible that the relationship between the Rhodomonas dnaX sequences and those of mycoplasmas is the result of long-branch attraction or a shared compositional bias. The role of dnaX in the Rhodomonas plastid is not known, but an obvious possibility is that it functions in DNA replication, perhaps interacting with or replacing some of the nucleus-encoded proteins that must be imported into the organelle. Experiments to better understand the distribution of dnaX in cryptophytes are currently underway.
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LGT is increasingly recognized as a major factor in plant mitochondrial genome evolution (e.g., Bergthorsson et al. 2003, 2004; Davis and Wurdack 2004
), yet LGTs involving plastid genes appear to be exceedingly rare. Indeed, a recent comprehensive survey of 204 plastid-encoded genes by Rice and Palmer (2006) revealed only 1 convincing case of LGT in addition to the previously characterized proteobacterial-derived large and small subunits of RuBisCO in red algal plastids and their derivatives (Delwiche and Palmer 1996). In this instance, a noncyanobacterial type rpl36 gene was identified in cryptophytes and haptophytes (Rice and Palmer 2006); the simplest interpretation is that the gene replaced the canonical cyanobacterial homolog in a common ancestor of cryptophytes and haptophytes after they diverged from heterokonts. The dnaX transfer presented here represents only the third plastid LGT to be documented and the second directly involving cryptophyte algae. The significance of this observation is not clear, although differences in the apparent frequency of mitochondrial and plastid LGTs have been linked to the presence/absence of DNA uptake systems (Rice and Palmer 2006). It is possible that cryptophyte plastids and, more generally, those of chromists and red algae are atypical in this regard. While single-gene phylogenies of all analyzable R. salina plastid proteins revealed no additional convincing cases of LGT beyond dnaX and rpl36 (data not shown), the presence of multiple apparently unrelated mobile genetic elements in different genomic contexts in the plastids of Rhodomonas species (Archibald Lab, unpublished) is consistent with the notion of enhanced DNA uptake in cryptophyte plastids.
Concatenated Protein Phylogenies and Chromist Plastid Evolution
While phylogenetic studies have demonstrably shown that cryptophyte, heterokont, and haptophyte plastids are derived from red algae (reviewed by Bhattacharya, Yoon, and Hackett 2003; Palmer 2003; Keeling 2004; Archibald and Keeling 2005), single- and multi-gene analyses of plastid proteins have produced conflicting results regarding their origin(s). With complete plastid genome sequences now available from 2 cryptophytes (this study; Douglas and Penny 1999), 3 heterokonts (Kowallik 1995; Oudot-Le Secq et al. 2007), a haptophyte (Puerta, Bachvaroff, and Delwiche 2005) and 4 red algae (Reith 1995; Glöckner, Rosenthal, and Valentin 2000; Ohta et al. 2003; Hagopian et al. 2004), we assembled a concatenate of 45 proteins encoded in 15 plastid genomes as well as homologs from 2 cyanobacteria (9,081 amino acids in total with no missing data). This dataset (and derivatives thereof) was subjected to a battery of phylogenetic analyses aimed at rigorously testing relationships within and between chromists and red algae. Due to the limited coding capacity of their plastid genomes (see Bachvaroff, Sanchez Puerta, and Delwiche 2005 and references therein), sequences from chlorophyll-c-containing dinoflagellates were not included.
The 45-protein dataset was first analyzed using PhyML (Guindon and Gascuel 2003) and IQPNNI (Vinh le and Von Haeseler 2004), 2 methods that explore tree space using maximum likelihood (ML), as well as PhyloBayes, a method employing a site-heterogeneous model to account for position-specific characteristics of protein evolution (Lartillot and Philippe 2004; Lartillot, Brinkmann, and Philippe 2007). All 3 methods recovered a monophyletic chromist assemblage with high support (fig. 3A, Supplementary figs. S1A–C). Heterokonts and haptophytes consistently branched as each other's closest relatives, as has been observed in recent analyses (Yoon et al. 2002; Rogers et al. 2007), and the chromists branched with the Gracilaria/Porphyra clade of red algae to the exclusion of the cyanidiales (fig. 3A). More generally, the position of the glaucophyte Cyanophora paradoxa relative to green algae/land plants and red algae/chromists varied depending on the method used, branching weakly with green plastids in the PhyloBayes tree. Removal of the fastest-evolving sites (Materials and Methods) had no effect on the resulting tree topologies, although statistical support for chromist monophyly decreased from 93 to 77% with PhyML (fig. 3A). Significantly, the improved chromist taxon sampling used here produced trees largely unaffected by the inclusion of proteins involved in transcription and translation in the full concatenate, unlike previous results (Martin et al. 1998; Hagopian et al. 2004). Anomalous results were, however, obtained in analyses of transcription and translation proteins on their own. For example, PhyloBayes analysis of a 16-protein transcription/translation-only dataset resulted in a topology in which cryptophytes and the haptophyte Emiliania huxleyi branched as sister taxa with a posterior probability of 0.97 (Supplementary fig. S1N; see below). Exclusion of the cyanidiales, which represent long branches in our trees and have been shown to be problematic in concatenated analyses of nuclear genes (Rodriguez-Ezpeleta et al. 2005), did not impact the relative branching order of the 3 chromist groups or support for chromist monophyly (Supplementary figs. S1Q–1T).
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We next tested the impact of protein composition bias on our concatenated phylogenies by recoding the amino acids into 4 groups with biochemically similar properties as done by Hrdy et al. (2004)
, coding cysteine as missing data and combining hydrophobic (MILV) and aromatic (FYW) amino acids as a single category (Rodriguez-Ezpeleta et al. 2007). Analysis of the 45-protein recoded dataset produced strikingly different results (fig. 3B) from those described above. While the chromists and red algae still branched together, support for chromist monophyly disintegrated. Cryptophytes and haptophytes branched weakly as sister groups, with the heterokonts branching outside a cluster of these taxa together with Porphyra and Gracilaria. When the fastest-evolving sites were removed from the recoded dataset, chromist monophyly was once again recovered, although support was still weak (Supplementary fig. S1H). A consistent observation in both the standard and recoded analyses was the long branches observed in the heterokont sequences as well as those of the cyanidiales.
The dramatic differences seen in analyses performed with and without amino acid recoding led us to explore the impact of protein composition bias further. Specifically, we calculated GARP/FYMINK ratios (Foster and Hickey 1999) for each protein and each taxon and assessed the between-taxon variation for each. The results indicate that a subset of the proteins in our concatenate exhibit significant variation in GARP/FYMINK ratios, with the cyanidiales, heterokonts, and single haptophyte being the outliers in most cases (Supplementary fig. S2A). Photosynthesis- and nonphotosynthesis-related proteins were distributed more or less randomly when proteins were ranked from lowest to highest in terms of standard deviation (SD). We performed analyses on subsets of the concatenate: 9 proteins with SD > 0.4, the remaining 36 proteins, and 21 proteins with SD < 0.2 (Supplementary Figs. S3A–S3L). Interestingly, the proteins with the most inter-taxon variation in GARP/FYMINK ratio usually produced topologies incongruent with the full concatenate, often with cryptophytes and haptophytes as sister taxa and with the heterokonts branching elsewhere in the tree (e.g., Supplementary fig. S2B). PhyloBayes was the only method that still recovered chromist monophyly when the most compositionally varied proteins were analyzed in isolation (Supplementary fig. S3B). This is consistent with the fact that the method appears resistant to systematic artifacts such as long-branch attraction (Lartillot, Brinkmann, and Philippe 2007).
Rice and Palmer (2006) recently described the presence of an LGT-derived noncyanobacterial-type rpl36 gene in the plastids of cryptophytes and haptophytes, which was interpreted as evidence for the sisterhood of these 2 groups to the exclusion of heterokonts and alveolates. This relationship is inconsistent with the bulk of the plastid phylogenies presented in this study and elsewhere (Yoon et al. 2002; Hagopian et al. 2004; Rogers et al. 2007). Nevertheless, the sisterhood of cryptophytes and haptophytes is observed in some of our analyses (e.g., fig. 3B, Supplementary fig. S1N) and recent phylogenies of nucleus-encoded proteins have provided support for a specific relationship between these 2 groups (Harper, Waanders, and Keeling 2005; Hackett et al. 2007; Patron, Inagaki, and Keeling 2007). We used AU tests to further assess the relative branching order within and between chromists and red algae using the PhyloBayes tree shown in figure 3A as a reference. From a set of 315 alternate trees, only 3 topologies were not rejected at P < 0.05. Significantly, these include the tree shown in figure 3B and 2 trees in which the red algae G. tenuistitipata and P. purpurea were placed as sister to the cryptophytes and the haptophytes and heterokonts were monophyletic. Our genome-wide single-gene/protein analyses of the R. salina genome revealed that, of the 103 proteins analyzed, a full 38% produced phylogenies in which cryptophytes and haptophytes were sister taxa, consistent with the results of Rice and Palmer (2006). Statistical support for this relationship was highly variable (<20%–100%; average = 48%), as expected from single-gene phylogenies inferred from anciently diverged sequences.
In an effort to shed light on the apparently contradictory scenarios for the evolutionary relationship amongst the 3 chromist lineages, we performed a site-by-site likelihood analysis of all 9,081 amino acids present in the 45-protein concatenate under 2 different topologies, one in which haptophytes and heterokonts were each others’ closest relatives (i.e., the topology shown in fig. 3A), the other where cryptophytes and haptophytes branch together, as predicted by the rpl36 LGT (Rice and Palmer 2006). The differences in log likelihood (
lnL) for each site were then plotted and ordered according to their inferred evolutionary rate (fig. 4). In total, 5,413 sites support a haptophyte-heterokont relationship, while 3,668 sites favor a cryptophyte-haptophyte relationship (fig. 4; the majority of the sites in the alignment [3,370] were invariant with very small lnL values and a negligible impact on support for one topology over the other). When considering sites belonging to site rate categories 2–8, 1,994 sites support a haptophyte-cryptophyte relationship, while 3,718 sites support a haptophyte-heterokont relationship. This result is consistent with the full-dataset concatenated phylogenies (figs. 3A, Supplementary figs. S1 and S2) that support a haptophyte-heterokont relationship. The only 2 groups of amino acid sites that support a haptophyte-cryptophyte relationship over heterokonts-haptophytes fall into site rate categories 7 and 8, i.e., the fast-evolving sites. As expected, removal of the fast-evolving sites from the full-concatenated dataset resulted in increased bootstrap support for the monophyly of haptophytes and heterokonts (Supplementary fig. S1), but decreased support for the node uniting chromists. The majority of the sites that support a haptophyte-heterokont relationship in site rate categories 2–8 have lnL values between 0 and –0.1 (3,063 sites), the same being the case for the haptophyte+cryptophyte relationship (1,342 sites between 0 and +0.1). Unexpectedly, the total number of sites with more extreme lnL values (i.e., > +0.1 or < –0.1) in support of the 2 topologies is approximately equal: 652 sites for haptophytes-cryptophytes, 655 for heterokonts-haptophytes (fig. 4). This indicates that the high statistical support for the haptophyte-heterokont relationship seen in the concatenated phylogenies is the result of the combined signal from a proportionately large number of amino acid positions (3,063) that do not strongly discriminate between the 2 topologies. The total number of sites that strongly support haptophytes+heterokonts versus cryptophytes+haptophytes is very similar.
|
As discussed above, 38% of the individual proteins analyzed in this study showed cryptophytes and haptophytes as each other's closest relatives. In order to identify individual proteins in support of the alternative topologies shown in figure 4, we performed a likelihood analysis of each of the 45 proteins analyzed in the full dataset in isolation. Consistent with the results described above, and a similar analysis performed by Rice and Palmer (2006)
, 28 out of 45 proteins (62%) supported a haptophyte-heterokont relationship (fig. S4). Nine genes/proteins (20%) possessed lnL values in the range of +0.033 to –0.05 (atpF, petG, rpl20, rps18, psbN, psbF, psbK, psbL, rps11), indicating that they support neither topology very strongly. With the exception of rpl20, all of the above-mentioned proteins have relatively high GARP/FYMINK ratios (Supplementary fig. S2A), and most are less than 100 amino acids in size. On the one extreme, the photosystem genes psbB, psaA, and petD strongly support haptophytes+heterokonts, as was shown to be the case previously (Yoon et al. 2002). On the other, rps14, rpoB, and atpB strongly favor cryptophytes+haptophytes. |
Conclusion |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionSuppementary MaterialAcknowledgementsReferences |
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We have completely sequenced the plastid genome of the cryptophyte alga Rhodomonas salina and identified the first known case of a putative DNA polymerase enzyme encoded in plastid DNA. Phylogenies constructed from a large concatenated set of plastid proteins provide support for the monophyly of chromist plastids and consistently favor a relationship between heterokonts and haptophytes to the exclusion of cryptophytes. However, we have also shown that support for the chromist plastid monophyly and heterokont-haptophyte sisterhood vanishes when the data is recoded to minimize the impact of amino acid composition bias, leaving open the possibility that an as yet undetermined systematic bias is responsible for the strongly supported trees presented herein. We can thus neither confirm nor refute a specific relationship between the plastids of cryptophytes and haptophytes as suggested by the shared presence of an LGT-derived rpl36 gene (Rice and Palmer 2006
) and recent large-scale analyses of nuclear genes (Hackett et al. 2007; Patron, Inagaki, and Keeling 2007). Additional plastid genome sequences from diverse haptophytes, heterokonts, and cryptophytes will be required to further break up the long terminal branches that presently characterize chromist plastid phylogenies. It is also important to consider that sequences from the fourth chlorophyll c–containing algal lineage, the dinoflagellates, were not included in our analysis, and complete plastid genomes are currently only available from a small fraction of the known diversity of red algae (Yoon et al. 2006). The impact of these missing data on the relative branching order of the 3 chromist lineages and, more generally, support for chromist monophyly is presently unclear. Together with trees inferred from concatenated nuclear gene sequences from chromists, dinoflagellates, and apicomplexans, analysis of a further expanded plastid protein dataset should make it possible to assess the congruence between the host cell and plastid components of these complex organisms and definitively test the hypothesis of a single endosymbiotic origin of plastids in chromist and chromalveolate taxa (Cavalier-Smith 1999). |
Suppementary Material |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionSuppementary MaterialAcknowledgementsReferences |
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Supplementary figures and table are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
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Acknowledgements |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionSuppementary MaterialAcknowledgementsReferences |
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We are grateful to Marie-Pierre Oudot-Le Secq and Beverly R. Green for providing access to the T. pseudonana and P. tricornutum plastid genomes before public release and to Jessica Leigh, Andrew Roger, Susan Douglas, and Gabino Sanchez Perez for help with analyses and for comments on the manuscript. Three anonymous reviewers are also thanked for helpful suggestions. This work was supported by Genome Atlantic and an operating grant awarded to J.M.A. from NSERC. J.M.A. is a Scholar of the Canadian Institute for Advanced Research, Program in Evolutionary Biology.
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Geoffrey McFadden, Associate Editor
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