A new scenario of plastid evolution: plastid primary endosymbiosis before the divergence of the “Plantae,” emended

A recent hypothesis on the origin of eukaryotic phototrophs proposes that red algae, green plants (land plants plus green algae), and glaucophytes constitute the primary photosynthetic eukaryotes, whose plastids may have originated directly from a cyanobacterium-like prokaryote via primary endosymbiosis, whereas the plastids of other lineages of eukaryotic phototrophs appear to be the result of secondary endosymbiotic events involving a phototrophic eukaryote and a host cell. However, the phylogenetic relationships among the three lineages of primary photosynthetic eukaryotes remained unresolved because previous nuclear multigene phylogenies used incomplete red algal gene sequences derived mainly from Porphyra (Rhodophyceae, one of the two lineages of the Rhodophyta), and lacked sequences from the Cyanidiophyceae (the other red algal lineage). Recently, the complete nuclear genome sequences from the red alga Cyanidioschyzon merolae 10D of the Cyanidiophyceae were determined. Using this genomic information, nuclear multigene phylogenetic analyses of various lineages of mitochondrion-containing eukaryotes were conducted. Since bacterial and amitochondrial eukaryotic genes present serious problems to eukaryotic phylogenies, basal eukaryotes were deduced based on the paralogous comparison of the concatenated - and -tubulin. The comparison demonstrated that cellular slime molds (Amoebozoa) represent the most basal position within the mitochondrion-containing organisms. With the cellular slime molds as the outgroup, phylogenetic analyses based on a 1,525-amino acid sequence of four concatenated nuclear genes [actin, elongation factor-1( EF-1), -tubulin, and -tubulin] resolved the presence of two large, robust monophyletic groups and the basal eukaryotic lineages (Amoebozoa). One of the two groups corresponded to the Opisthokonta (Metazoa and Fungi), whereas the other included various lineages containing primary and secondary plastids (red algae, green plants, glaucophytes, euglenoids, heterokonts, and apicomplexans), Ciliophora, Kinetoplastida, dinoflagellates, and Heterolobosea, for which the red algae represented the most basal lineage. Therefore, the plastid primary endosymbiosis likely occurred once in the common ancestor of the latter group, and the primary plastids were subsequently lost in the ancestor(s) of organisms within the group that now lacks primary plastids. A new concept of Plantae was proposed for phototrophic and nonphototrophic organisms belonging to this group on the basis of their common history of plastid primary endosymbiosis. This new scenario of plastid evolution is discussed here, and is compared with recent genome information and findings on the secondary endosymbiosis of the Euglena plastid.     

Segregate or cooperate- a study of the interaction between two species of Dictyostelium

Background

Plastids have inherited their own genomes from a single cyanobacterial ancestor, but the majority of cyanobacterial genes, once retained in the ancestral plastid genome, have been lost or transferred into the eukaryotic host nuclear genome via endosymbiotic gene transfer. Although previous studies showed that cyanobacterial gnd genes, which encode 6-phosphogluconate dehydrogenase, are present in several plastid-lacking protists as well as primary and secondary plastid-containing phototrophic eukaryotes, the evolutionary paths of these genes remain elusive.

Results

Here we show an extended phylogenetic analysis including novel gnd gene sequences from Excavata and Glaucophyta. Our analysis demonstrated the patchy distribution of the excavate genes in the gnd gene phylogeny. The Diplonema gene was related to cytosol-type genes in red algae and Opisthokonta, while heterolobosean genes occupied basal phylogenetic positions with plastid-type red algal genes within the monophyletic eukaryotic group that is sister to cyanobacterial genes. Statistical tests based on exhaustive maximum likelihood analyses strongly rejected that heterolobosean gnd genes were derived from a secondary plastid of green lineage. In addition, the cyanobacterial gnd genes from phototrophic and phagotrophic species in Euglenida were robustly monophyletic with Stramenopiles, and this monophyletic clade was moderately separated from those of red algae. These data suggest that these secondary phototrophic groups might have acquired the cyanobacterial genes independently of secondary endosymbioses.

Conclusion

We propose an evolutionary scenario in which plastid-lacking Excavata acquired cyanobacterial gnd genes via eukaryote-to-eukaryote lateral gene transfer or primary endosymbiotic gene transfer early in eukaryotic evolution, and then lost either their pre-existing or cyanobacterial gene.     

The phylogenetic position of red algae revealed by multiple nuclear genes from mitochondria-containing eukaryotes and an alternative hypothesis on the origin of plastids

Abstract Red algae are one of the main photosynthetic eukaryotic lineages and are characterized by primitive features, such as a lack of flagella and the presence of phycobiliproteins in the chloroplast. Recent molecular phylogenetic studies using nuclear gene sequences suggest two conflicting hypotheses (monophyly versus non-monophyly) regarding the relationships between red algae and green plants. Although kingdom-level phylogenetic analyses using multiple nuclear genes from a wide-range of eukaryotic lineages were very recently carried out, they used highly divergent gene sequences of the cryptomonad nucleomorph (as the red algal taxon) or incomplete red algal gene sequences. In addition, previous eukaryotic phylogenies based on nuclear genes generally included very distant archaebacterial sequences (designated as the outgroup) and/or amitochondrial organisms, which may carry unusual gene substitutions due to parasitism or the absence of mitochondria. Here, we carried out phylogenetic analyses of various lineages of mitochondria-containing eukaryotic organisms using nuclear multigene sequences, including the complete sequences from the primitive red alga Cyanidioschyzon merolae. Amino acid sequence data for two concatenated paralogous genes (α- and β-tubulin) from mitochondria-containing organisms robustly resolved the basal position of the cellular slime molds, which were designated as the outgroup in our phylogenetic analyses. Phylogenetic analyses of 53 operational taxonomic units (OTUs) based on a 1525-amino-acid sequence of four concatenated nuclear genes (actin, elongation factor-1α, α-tubulin, and β-tubulin) reliably resolved the phylogeny only in the maximum parsimonious (MP) analysis, which indicated the presence of two large robust monophyletic groups (Groups A and B) and the basal eukaryotic lineages (red algae, true slime molds, and amoebae). Group A corresponded to the Opisthokonta (Metazoa and Fungi), whereas Group B included various primary and secondary plastid-containing lineages (green plants, glaucophytes, euglenoids, heterokonts, and apicomplexans), Ciliophora, Kinetoplastida, and Heterolobosea. The red algae represented the sister lineage to Group B. Using 34 OTUs for which essentially the entire amino acid sequences of the four genes are known, MP, distance, quartet puzzling, and two types of maximum likelihood (ML) calculations all robustly resolved the monophyly of Group B, as well as the basal position of red algae within eukaryotic organisms. In addition, phylogenetic analyses of a concatenated 4639-amino-acid sequence for 12 nuclear genes (excluding the EF-2 gene) of 12 mitochondria-containing OTUs (including C. merolae) resolved a robust non-sister relationship between green plants and red algae within a robust monophyletic group composed of red algae and the eukaryotic organisms belonging to Group B. A new scenario for the origin and evolution of plastids is suggested, based on the basal phylogenetic position of the red algae within the large clade (Group B plus red algae). The primary plastid endosymbiosis likely occurred once in the common ancestor of this large clade, and the primary plastids were subsequently lost in the ancestor(s) of the Discicristata (euglenoids, Kinetoplastida, and Heterolobosea), Heterokontophyta, and Alveolata (apicomplexans and Ciliophora). In addition, a new concept of “Plantae” is proposed for phototrophic and nonphototrophic organisms belonging to Group B and red algae, on the basis of the common history of the primary plastid endosymbiosis. The Plantae include primary plastid-containing phototrophs and nonphototrophic eukaryotes that possibly contain genes of cyanobacterial origin acquired in the primary endosymbiosis.     

Phylogeny of Plastids Based on Cladistic Analysis of Gene Loss Inferred from Complete Plastid Genome Sequences

Based on the recent hypothesis on the origin of eukaryotic phototrophs, red algae, green plants, and glaucophytes constitute the primary photosynthetic eukaryotes (whose plastids may have originated directly from a cyanobacterium-like prokaryote via primary endosymbiosis), whereas the plastids of other lineages of eukaryotic phototrophs appear to be the result of secondary or tertiary endosymbiotic events (involving a phototrophic eukaryote and a host cell). Although phylogenetic analyses using multiple plastid genes from a wide range of eukaryotic lineages have been carried out, some of the major phylogenetic relationships of plastids remain ambiguous or conflict between different phylogenetic methods used for nucleotide or amino acid substitutions. Therefore, an alternative methodology to infer the plastid phylogeny is needed. Here, we carried out a cladistic analysis of the loss of plastid genes after primary endosymbiosis using complete plastid genome sequences from a wide range of eukaryotic phototrophs. Since it is extremely unlikely that plastid genes are regained during plastid evolution, we used the irreversible Camin-Sokal model for our cladistic analysis of the loss of plastid genes. The cladistic analysis of the 274 plastid protein-coding genes resolved the 20 operational taxonomic units representing a wide range of eukaryotic lineages (including three secondary plastid-containing groups) into two large monophyletic groups with high bootstrap values: one corresponded to the red lineage and the other consisted of a large clade composed of the green lineage (green plants and Euglena) and the basal glaucophyte plastid. Although the sister relationship between the green lineage and the Glaucophyta was not resolved in recent phylogenetic studies using amino acid substitutions from multiple plastid genes, it is consistent with the rbcL gene phylogeny and with a recent phylogenetic study using multiple nuclear genes. In addition, our analysis robustly resolved the conflicting/ambiguous phylogenetic positions of secondary plastids in previous phylogenetic studies: the Euglena plastid was sister to the chlorophycean (Chlamydomonas) lineage, and the secondary plastids from the diatom (Odontiella) and cryptophyte (Guillardia) were monophyletic within the red lineage.     

A tertiary plastid uses genes from two endosymbionts

The origin and subsequent spread of plastids by endosymbiosis had a major environmental impact and altered the course of a great proportion of eukaryotic biodiversity. The ancestor of dinoflagellates contained a secondary plastid that was acquired in an ancient endosymbiotic event, where a eukaryotic cell engulfed a red alga. This is known as secondary endosymbiosis and has happened several times in eukaryotic evolution. Certain dinoflagellates, however, are unique in having replaced this secondary plastid in an additional (tertiary) round of endosymbiosis. Most plastid proteins are encoded in the nucleus of the host and are targeted to the organelle. When secondary or tertiary endosymbiosis takes place, it is thought that these genes move from nucleus to nucleus, so the plastid retains the same proteome. We have conducted large-scale expressed sequence tag (EST) surveys from Karlodinium micrum, a dinoflagellate with a tertiary haptophyte-derived plastid, and two haptophytes, Isochrysis galbana and Pavlova lutheri. We have identified all plastid-targeted proteins, analysed the phylogenetic origin of each protein, and compared their plastid-targeting transit peptides. Many plastid-targeted genes in the Karlodinium nucleus are indeed of haptophyte origin, but some genes were also retained from the original plastid (showing the two plastids likely co-existed in the same cell), in other cases multiple isoforms of different origins exist. We analysed plastid-targeting sequences and found the transit peptides in K.micrum are different from those found in either dinoflagellates or haptophytes, pointing to a plastid with an evolutionarily chimeric proteome, and a massive remodelling of protein trafficking during plastid replacement.     

Tightly Constrained Genome Reduction and Relaxation of Purifying Selection during Secondary Plastid Endosymbiosis

Endosymbiosis, the establishment of a former free-living prokaryotic or eukaryotic cell as an organelle inside a host cell, can dramatically alter the genomic architecture of the endosymbiont. Plastids or chloroplasts, the light-harvesting organelle of photosynthetic eukaryotes, are excellent models to study this phenomenon because plastid origin has occurred multiple times in evolution. Here, we investigate the genomic signature of molecular processes acting through secondary plastid endosymbiosis—the origination of a new plastid from a free-living eukaryotic alga. We used phylogenetic comparative methods to study gene loss and changes in selective regimes on plastid genomes, focusing on green algae that have given rise to three independent lineages with secondary plastids (euglenophytes, chlorarachniophytes, and Lepidodinium). Our results show an overall increase in gene loss associated with secondary endosymbiosis, but this loss is tightly constrained by the retention of genes essential for plastid function. The data show that secondary plastids have experienced temporary relaxation of purifying selection during secondary endosymbiosis. However, this process is tightly constrained, with selection relaxed only relative to the background in primary plastids. Purifying selection remains strong in absolute terms even during the endosymbiosis events. Selection intensity rebounds to pre-endosymbiosis levels following endosymbiosis events, demonstrating the changes in selection efficiency during different origin phases of secondary plastids. Independent endosymbiosis events in the euglenophytes, chlorarachniophytes, and Lepidodinium differ in their degree of relaxation of selection, highlighting the different evolutionary contexts of these events. This study reveals the selection–drift interplay during secondary endosymbiosis and evolutionary parallels during organellogenesis.     

The endosymbiotic origin,diversification and fate of plastids

Plastids and mitochondria each arose from a single endosymbiotic event and share many similarities in how they were reduced and integrated with their host. However, the subsequent evolution of the two organelles could hardly be more different: mitochondria are a stable fixture of eukaryotic cells that are neither lost nor shuffled between lineages, whereas plastid evolution has been a complex mix of movement, loss and replacement. Molecular data from the past decade have substantially untangled this complex history, and we now know that plastids are derived from a single endosymbiotic event in the ancestor of glaucophytes, red algae and green algae (including plants). The plastids of both red algae and green algae were subsequently transferred to other lineages by secondary endosymbiosis. Green algal plastids were taken up by euglenids and chlorarachniophytes, as well as one small group of dinoflagellates. Red algae appear to have been taken up only once, giving rise to a diverse group called chromalveolates. Additional layers of complexity come from plastid loss, which has happened at least once and probably many times, and replacement. Plastid loss is difficult to prove, and cryptic, non-photosynthetic plastids are being found in many non-photosynthetic lineages. In other cases, photosynthetic lineages are now understood to have evolved from ancestors with a plastid of different origin, so an ancestral plastid has been replaced with a new one. Such replacement has taken place in several dinoflagellates (by tertiary endosymbiosis with other chromalveolates or serial secondary endosymbiosis with a green alga), and apparently also in two rhizarian lineages: chlorarachniophytes and Paulinella (which appear to have evolved from chromalveolate ancestors). The many twists and turns of plastid evolution each represent major evolutionary transitions, and each offers a glimpse into how genomes evolve and how cells integrate through gene transfers and protein trafficking.     

PRIMARY AND SECONDARY ENDOSYMBIOSIS AND THE ORIGIN OF PLASTIDS

The theory of endosymbiosis describes the origin of plastids from cyanobacterial-like prokaryotes living within eukaryotic host cells. The endosymbionts are much reduced, but morphological, biochemical, and molecular studies provide clear evidence of a prokaryotic ancestry for plastids. There appears to have been a single (primary) endosymbiosis that produced plastids with two bounding membranes, such as those in green algae, plants, red algae, and glaucophytes. A subsequent round of endosymbioses, in which red or green algae were engulfed and retained by eukaryotic hosts, transferred photosynthesis into other eukaryotic lineages. These endosymbiotic plastid acquisitions from eukaryotic algae are referred to as secondary endosymbioses, and the resulting plastids classically have three or four bounding membranes. Secondary endosymbioses have been a potent factor in eukaryotic evolution, producing much of the modern diversity of life.     

Synchroma grande spec. nov. (Synchromophyceae class. nov., Heterokontophyta): an amoeboid marine alga with unique plastid complexes

Chromist algae including the Heterokontophyta are supposed to have evolved monophyletically by secondary endosymbiosis from a eukaryotic host cell that engulfed a eukaryotic red alga. The red algal endosymbiont was then reduced to a secondary plastid surrounded by four enveloping membranes. On the basis of the amoeboid marine alga Synchroma grande gen. et spec. nov., the Synchromophyceae are described here as a new class of Heterokontophyta. Their taxonomic position is characterized by 18S rRNA and rbcL gene phylogenies, morphology, and pigment composition. The so far unique feature of the Synchromophyceae is the occurrence of conspicuous chloroplast complexes representing multiplastidic red secondary endosymbionts. In these remarkable secondary endosymbionts, several primary chloroplasts are aggregated in a common periplastidial compartment and are collectively enveloped by an additional outer membrane pair. The discovery of this novel plastid morphology is highly relevant for research on algal evolution and is discussed in terms of the postulated monophyletic origin of Chromista.     

THE MESOZOIC RADIATION OF EUKARYOTIC ALGAE: THE PORTABLE PLASTID HYPOTHESIS1

Although all chloroplasts appear to have been derived from a common ancestor, a major schism occurred early in the evolution of eukaryotic algae that gave rise to red and green photoautotrophic lineages. In Paleozoic and earlier times, the fossil record suggests that oceanic eukaryotic phytoplankton were dominated by the green (chl b‐containing) algal line. However, following the end‐Permian extinction, a diverse group of eukaryotic phytoplankton evolved from secondary symbiotic associations in the red (chl c‐containing) line and subsequently rose to ecological prominence. In the contemporary oceans, red eukaryotic phytoplankton taxa continue to dominate marine pelagic food webs, whereas the green line is relegated to comparatively minor ecological and biogeochemical roles. To help elucidate why the oceans are not dominated by green taxa, we analyzed and compared whole plastid genomes in both the red and green lineages. Our results suggest that whereas all algal plastids retain a core set of genes, red plastids retain a complementary set of genes that potentially confer more capacity to autonomously express proteins regulating oxygenic photosynthetic and energy transduction pathways. We hypothesize that specific gene losses in the primary endosymbiotic green plastid reduced its portability for subsequent symbiotic associations. This corollary of the plastid “enslavement” hypothesis may have limited subsequent evolutionary advances in the green lineage while simultaneously providing a competitive advantage to the red lineage.     

A Genomic and Phylogenetic Perspective on Endosymbiosis and Algal Origin

Accounting for the diversity of photosynthetic eukaryotes is an important challenge in microbial biology. It has now become clear that endosymbiosis explains the origin of the photosynthetic organelle (plastid) in different algal groups. The first plastid originated from a primary endosymbiosis, whereby a previously non-photosynthetic protist engulfed and enslaved a cyanobacterium. This alga then gave rise to the red, green, and glaucophyte lineages. Algae such as the chlorophyll c-containing chromists gained their plastid through secondary endosymbiosis, in which an existing eukaryotic alga (in this case, a rhodophyte) was engulfed. Another chlorophyll c-containing algal group, the dinoflagellates, is a member of the alveolates that is postulated to be sister to chromists. The plastid in these algae has followed a radically different path of evolution. The peridinin-containing dinoflagellates underwent an unprecedented level of plastid genome reduction with the ca. 16 remaining genes encoded on 1–3 gene minicircles. In this short review, we examine algal plastid diversity using phylogenetic and genomic methods and show endosymbiosis to be a major force in algal evolution. In particular, we focus on the evolution of targeting signals that facilitate the import of nuclear-encoded photosynthetic proteins into the plastid.     

Phylogeny and Self-Splicing Ability of the Plastid tRNA-Leu Group I Intron

Group I introns are mobile RNA enzymes (ribozymes) that encode conserved primary and secondary structures required for autocatalysis. The group I intron that interrupts the tRNA-Leu gene in cyanobacteria and plastids is remarkable because it is the oldest known intervening sequence and may have been present in the common ancestor of the cyanobacteria (i.e., 2.7–3.5 billion years old). This intron entered the eukaryotic domain through primary plastid endosymbiosis. We reconstructed the phylogeny of the tRNA-Leu intron and tested the in vitro self-splicing ability of a diverse collection of these ribozymes to address the relationship between intron stability and autocatalysis. Our results suggest that the present-day intron distribution in plastids is best explained by strict vertical transmission, with no intron losses in land plants or a subset of the Stramenopiles (xanthophyceae/phaeophyceae) and frequent loss among green algae, as well as in the red algae and their secondary plastid derivatives (except the xanthophyceae/phaeophyceae lineage). Interestingly, all tested land plant introns could not self-splice in vitro and presumably have become dependent on a host factor to facilitate in vivo excision. The host dependence likely evolved once in the common ancestor of land plants. In all other plastid lineages, these ribozymes could either self-splice or complete only the first step of autocatalysis. The first two authors (Dawn Simon and David Fewer) have contributed equally to this work. Present address (David Fewer): Department of Applied Chemistry and Microbiology, Viikki Biocenter, P.O. Box 56, Viikinkaari 9, 00014 University of Helsinki, Helsinki, Finland     

A “Green” Phosphoribulokinase in Complex Algae with Red Plastids: Evidence for a Single Secondary Endosymbiosis Leading to Haptophytes, Cryptophytes, Heterokonts, and Dinoflagellates

Phosphoribulokinase (PRK) is an essential enzyme of photosynthetic eukaryotes which is active in the plastid-located Calvin cycle and regenerates the substrate for ribulose-bisphosphate carboxylase/oxygenase (Rubisco). Rhodophytes and chlorophytes (red and green algae) recruited their nuclear-encoded PRK from the cyanobacterial ancestor of plastids. The plastids of these organisms can be traced back to a single primary endosymbiosis, whereas, for example, haptophytes, dinoflagellates, and euglenophytes obtained their “complex” plastids through secondary endosymbioses, comprising the engulfment of a unicellular red or green alga by a eukaryotic host cell. We have cloned eight new PRK sequences from complex algae as well as a rhodophyte in order to investigate their evolutionary origin. All available PRK sequences were used for phylogenetic analyses and the significance of alternative topologies was estimated by the approximately unbiased test. Our analyses led to several astonishing findings. First, the close relationship of PRK genes of haptophytes, heterokontophytes, cryptophytes, and dinophytes (complex red lineage) supports a monophyletic origin of their sequences and hence their plastids. Second, based on PRK genes the complex red lineage forms a highly supported assemblage together with chlorophytes and land plants, to the exclusion of the rhodophytes. This green affinity is in striking contrast to the expected red algal origin and our analyses suggest that the PRK gene was acquired once via lateral transfer from a green alga. Third, surprisingly the complex green lineages leading to Bigelowiella and Euglena probably also obtained their PRK genes via lateral gene transfers from a red alga and a complex alga with red plastids, respectively. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Patrick Keeling ] The nucleotide sequence data will appear in the DDBJ/EMBL/GenBank International Nucleotide Sequence Database under the following accession numbers. cDNA clones: AY772245 (Pavlova lutheri); AY772246 (Guillardia theta); AY772247 (Lingulodinium polyedrum); AY772248 and AY772249 (Pyrocystis lunula); AY772250 (Euglena gracilis); AY772251 (Chondrus crispus). Genomic clone: AY772252 (Prymnesium parvum). Genomic PCR clone: AY772253 (Bigelowiella natans).     

EVOLUTIONARY ANALYSES OF THE NUCLEAR‐ENCODED PHOTOSYNTHETIC GENE psbO FROM TERTIARY PLASTID‐CONTAINING ALGAE IN DINOPHYTA1

Although the dinophytes generally possess red‐algal‐derived secondary plastids, tertiary plastids originating from haptophyte and diatom ancestors are recognized in some lineages within the Dinophyta. However, little is known about the nuclear‐encoded genes of plastid‐targeted proteins from the dinophytes with diatom‐derived tertiary plastids. We analyzed the sequences of the nuclear psbO gene encoding oxygen‐evolving enhancer protein from various algae with red‐algal‐derived secondary and tertiary plastids. Based on our sequencing of 10 new genes and phylogenetic analysis of PsbO amino acid sequences from a wide taxon sampling of red algae and organisms with red‐algal‐derived plastids, dinophytes form three separate lineages: one composed of peridinin‐containing species with secondary plastids, and the other two having haptophyte‐ or diatom‐derived tertiary plastids and forming a robust monophyletic group with haptophytes and diatoms, respectively. Comparison of the N‐terminal sequences of PsbO proteins suggests that psbO genes from a dinophyte with diatom‐derived tertiary plastids (Kryptoperidinium) encode proteins that are targeted to the diatom plastid from the endosymbiotic diatom nucleus as in the secondary phototrophs, whereas the fucoxanthin‐containing dinophytes (Karenia and Karlodinium) have evolved an additional system of psbO genes for targeting the PsbO proteins to their haptophyte‐derived tertiary plastids from the host dinophyte nuclei.     

Evolution of P2A and P5A ATPases: ancient gene duplications and the red algal connection to green plants revisited

In a search for slowly evolving nuclear genes that may cast light on the deep evolution of plants, we carried out phylogenetic analyses of two well-characterized subfamilies of P-type pumps (P2A and P5A ATPases) from representative branches of the eukaryotic tree of life. Both P-type ATPase genes were duplicated very early in eukaryotic evolution and before the divergence of the present eukaryotic supergroups. Synapomorphies identified in the sequences provide evidence that green plants and red algae are more distantly related than are green plants and eukaryotic supergroups in which secondary or tertiary plastids are common, such as several groups belonging to the clade that includes Stramenopiles, Alveolata, Rhizaria, Cryptophyta and Haptophyta (SAR). We propose that red algae branched off soon after the first photosynthesizing eukaryote had acquired a primary plastid, while in another lineage that led to SAR, the primary plastid was lost but, in some cases, regained as a secondary or tertiary plastid.     

Mechanism of Protein Targeting to the Chlorarachniophyte Plastids and the Evolution of Complex Plastids with Four Membranes — A Hypothesis

Chlorarachniophyta are phototrophic amoeboflagellates, with plastids surrounded by four membranes. Contrary to other plastids of this type which occur in chromists, their outermost membrane bears no ribosomes. It is argued that the nuclear-encoded chlorarachniophyte plastid proteins are first transported into the ER, then to the Colgi apparatus, and finally to the plastids. The same import mechanism could be originally present in the chromist ancestor, prior to the fusion of their plastids with the RER membranes. According to the most recent concept, the complex plastids of Chromista and Chlorarachniophyta have evolved through replacement of the cyanobacterial plastids. The assumption that these plastids had an envelope composed not of two, but of three membranes makes it possible to avoid the erlier discerned difficulties with conversion of a eukaryotic alga into a complex plastid. My scenario provides an additional support to the hypothesis on polyphy-letic origin of four-membraned plastids.     

Multiple gene phylogenies support the monophyly of cryptomonad and haptophyte host lineages

Cryptomonad algae acquired their plastids by the secondary endosymbiotic uptake of a eukaryotic red alga. Several other algal lineages acquired plastids through such an event [1], but cryptomonads are distinguished by the retention of a relic red algal nucleus, the nucleomorph [2]. The nucleomorph (and its absence in other lineages) can reveal a great deal about the process and history of endosymbiosis, but only if we know the relationship between cryptomonads and other algae, and this has been controversial. Several recent analyses have suggested a relationship between plastids of cryptomonads and some or all other red alga-containing lineages [3-6], but we must also know whether host nuclear genes mirror this relationship to determine the number of endosymbiotic events, and this has not been demonstrated. We have carried out an expressed sequence tag (EST) survey of the cryptomonad Guillardia theta. Phylogenetic analyses of 102 orthologous nucleus-encoded proteins (18,425 amino acid alignment positions) show a robust sister-group relationship between cryptomonads and the haptophyte algae, which also have a red secondary plastid. This relationship demonstrates that loss of nucleomorphs must have taken place in haptophytes independently of any other red alga-containing lineages and that the ancestor of both already contained a red algal endosymbiont.     

Origins of the secondary plastids of Euglenophyta and Chlorarachniophyta as revealed by an analysis of the plastid‐targeting,nuclear‐encoded gene psbO1

Because the secondary plastids of the Euglenophyta and Chlorarachniophyta are very similar to green plant plastids in their pigment composition, it is generally considered that ancestral green algae were engulfed by other eukaryotic host cells to become the plastids of these two algal divisions. Recent molecular phylogenetic studies have attempted to resolve the phylogenetic positions of these plastids; however, almost all of the studies analyzed only plastid‐encoded genes. This limitation may affect the results of comparisons between genes from primary and secondary plastids, because genes in endosymbionts have a higher mutation rate than the genes of their host cells. Thus, the phylogeny of these secondary plastids must be elucidated using other molecular markers. Here, we compared the plastid‐targeting, nuclear‐encoded, oxygen‐evolving enhancer (psbO) genes from various green plants, the Euglenophyta and Chlorarachniophyta. A phylogenetic analysis based on the PsbO amino acid sequences indicated that the chlorarachniophyte plastids are positioned within the Chlorophyta (including Ulvophyceae, Chlorophyceae, and Prasinophyceae, but excluding Mesostigma). In contrast, plastids of the Euglenophyta and Mesostigma are positioned outside the Chlorophyta and Streptophyta. The relationship of these three phylogenetic groups was consistent with the grouping of the primary structures of the thylakoid‐targeting domain and its adjacent amino acids in the PsbO N‐terminal sequences. Furthermore, the serine‐X‐alanine (SXA) motif of PsbO was exactly the same in the Chlorarachniophyta and the prasinophycean Tetraselmis. Therefore, the chlorarachniophyte secondary plastids likely evolved from the ancestral Tetraselmis‐like alga within the Chlorophyta, whereas the Euglenophyte plastids may have originated from the unknown basal lineage of green plants.