Twenty-fold difference in evolutionary rates between the mitochondrial and plastid genomes of species with secondary red plastids

Within plastid-bearing species, the relative rates of evolution between mitochondrial and plastid genomes are poorly studied, but for the few lineages in which they have been explored, including land plants and green algae, the mitochondrial DNA mutation rate is nearly always estimated to be lower than or equal to that of the plastid DNA. Here, we show that in protists from three distinct lineages with secondary, red algal-derived plastids, the opposite is true: their mitochondrial genomes are evolving 5-30 times faster than their plastid genomes, even when the plastid is nonphotosynthetic. These findings have implications for understanding the origins and evolution of organelle genome architecture and the genes they encode.     

Nucleus-encoded,plastid-targeted glyceraldehyde-3-phosphate dehydrogenase (GAPDH) indicates a single origin for chromalveolate plastids

Plastids (the photosynthetic organelles of plants and algae) originated through endosymbiosis between a cyanobacterium and a eukaryote and subsequently spread to other eukaryotes by secondary endosymbioses between two eukaryotes. Mounting evidence favors a single origin for plastids of apicomplexans, cryptophytes, dinoflagellates, haptophytes, and heterokonts (together with their nonphotosynthetic relatives, termed chromalveolates), but so far, no single molecular marker has been described that supports this common origin. One piece of evidence comes from plastid-targeted glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which originated by a gene duplication of the cytosolic form. However, no plastid GAPDH has been characterized from haptophytes, leaving an important piece of the puzzle missing. We have sequenced genes encoding cytosolic, mitochondrion-targeted, and plastid-targeted GAPDH proteins from a number of haptophytes and heterokonts and found haptophyte homologs that branch within a strongly supported clade of chromalveolate plastid-targeted genes, being more closely related to an apicomplexan homolog than was expected. The evolution of plastid-targeted GAPDH supports red algal ancestry of apicomplexan plastids and raises a number of questions about the importance of plastid loss and the possibility of cryptic plastids in nonphotosynthetic lineages such as ciliates.     

Compartment-specific isoforms of TPI and GAPDH are imported into diatom mitochondria as a fusion protein: evidence in favor of a mitochondrial origin of the eukaryotic glycolytic pathway

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triosephosphate isomerase (TPI) are essential to glycolysis, the major route of carbohydrate breakdown in eukaryotes. In animals and other heterotrophic eukaryotes, both enzymes are localized in the cytosol; in photosynthetic eukaryotes, GAPDH and TPI exist as isoenzymes that function in the glycolytic pathway of the cytosol and in the Calvin cycle of chloroplasts. Here, we show that diatoms--photosynthetic protists that acquired their plastids through secondary symbiotic engulfment of a eukaryotic rhodophyte--possess an additional isoenzyme each of both GAPDH and TPI. Surprisingly, these new forms are expressed as an TPI-GAPDH fusion protein which is imported into mitochondria prior to its assembly into a tetrameric bifunctional enzyme complex. Homologs of this translational fusion are shown to be conserved and expressed also in nonphotosynthetic, heterokont-flagellated oomycetes. Phylogenetic analyses show that mitochondrial GAPDH and its N-terminal TPI fusion branch deeply within their respective eukaryotic protein phylogenies, suggesting that diatom mitochondria may have retained an ancestral state of glycolytic compartmentation that existed at the onset of mitochondrial symbiosis. These findings strongly support the view that nuclear genes for enzymes of glycolysis in eukaryotes were acquired from mitochondrial genomes and provide new insights into the evolutionary history (host-symbiont relationships) of diatoms and other heterokont-flagellated protists.     

Cyanobacterial contribution to the genomes of the plastid-lacking protists

Background  

Eukaryotic genes with cyanobacterial ancestry in plastid-lacking protists have been regarded as important evolutionary markers implicating the presence of plastids in the early evolution of eukaryotes. Although recent genomic surveys demonstrated the presence of cyanobacterial and algal ancestry genes in the genomes of plastid-lacking protists, comparative analyses on the origin and distribution of those genes are still limited.     

Nuclear-encoded, plastid-targeted genes suggest a single common origin for apicomplexan and dinoflagellate plastids

The phylum Apicomplexa encompasses a large number of intracellular protozoan parasites, including the causative agents of malaria (Plasmodium), toxoplasmosis (Toxoplasma), and many other human and animal diseases. Apicomplexa have recently been found to contain a relic, nonphotosynthetic plastid that has attracted considerable interest as a possible target for therapeutics. This plastid is known to have been acquired by secondary endosymbiosis, but when this occurred and from which type of alga it was acquired remain uncertain. Based on the molecular phylogeny of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes, we provide evidence that the apicomplexan plastid is homologous to plastids found in dinoflagellates-close relatives of apicomplexa that contain secondary plastids of red algal origin. Surprisingly, apicomplexan and dinoflagellate plastid-targeted GAPDH sequences were also found to be closely related to the plastid-targeted GAPDH genes of heterokonts and cryptomonads, two other groups that contain secondary plastids of red algal origin. These results address several outstanding issues: (1) apicomplexan and dinoflagellate plastids appear to be the result of a single endosymbiotic event which occurred relatively early in eukaryotic evolution, also giving rise to the plastids of heterokonts and perhaps cryptomonads; (2) apicomplexan plastids are derived from a red algal ancestor; and (3) the ancestral state of apicomplexan parasites was photosynthetic.     

Functional replacement of a primary metabolic pathway via multiple independent eukaryote‐to‐eukaryote gene transfers and selective retention

Although lateral gene transfer (LGT) is now recognized as a major force in the evolution of prokaryotes, the contribution of LGT to the evolution and diversification of eukaryotes is less understood. Notably, transfers of complete pathways are believed to be less likely between eukaryotes, because the successful transfer of a pathway requires the physical clustering of functionally related genes. Here, we report that in one of the closest unicellular relatives of animals, the choanoflagellate, Monosiga, three genes whose products work together in the glutamate synthase cycle are of algal origin. The concerted retention of these three independently acquired genes is best explained as the consequence of a series of adaptive replacement events. More generally, this study argues that (i) eukaryote‐to‐eukaryote transfers of entire metabolic pathways are possible, (ii) adaptive functional replacements of primary pathways can occur, and (iii) functional replacements involving eukaryotic genes are likely to have also contributed to the evolution of eukaryotes. Lastly, these data underscore the potential contribution of algal genes to the evolution of nonphotosynthetic lineages.     

Identification of plant-like galactolipids in Chromera velia, a photosynthetic relative of malaria parasites

Apicomplexa are protist parasites that include Plasmodium spp., the causative agents of malaria, and Toxoplasma gondii, responsible for toxoplasmosis. Most Apicomplexa possess a relict plastid, the apicoplast, which was acquired by secondary endosymbiosis of a red alga. Despite being nonphotosynthetic, the apicoplast is otherwise metabolically similar to algal and plant plastids and is essential for parasite survival. Previous studies of Toxoplasma gondii identified membrane lipids with some structural features of plastid galactolipids, the major plastid lipid class. However, direct evidence for the plant-like enzymes responsible for galactolipid synthesis in Apicomplexan parasites has not been obtained. Chromera velia is an Apicomplexan relative recently discovered in Australian corals. C. velia retains a photosynthetic plastid, providing a unique model to study the evolution of the apicoplast. Here, we report the unambiguous presence of plant-like monogalactosyldiacylglycerol and digalactosyldiacylglycerol in C. velia and localize digalactosyldiacylglycerol to the plastid. We also provide evidence for a plant-like biosynthesis pathway and identify candidate galactosyltranferases responsible for galactolipid synthesis. Our study provides new insights in the evolution of these important enzymes in plastid-containing eukaryotes and will help reconstruct the evolution of glycerolipid metabolism in important parasites such as Plasmodium and Toxoplasma.     

Nonessential plastid-encoded ribosomal proteins in tobacco: a developmental role for plastid translation and implications for reductive genome evolution

Plastid genomes of higher plants contain a conserved set of ribosomal protein genes. Although plastid translational activity is essential for cell survival in tobacco (Nicotiana tabacum), individual plastid ribosomal proteins can be nonessential. Candidates for nonessential plastid ribosomal proteins are ribosomal proteins identified as nonessential in bacteria and those whose genes were lost from the highly reduced plastid genomes of nonphotosynthetic plastid-bearing lineages (parasitic plants, apicomplexan protozoa). Here we report the reverse genetic analysis of seven plastid-encoded ribosomal proteins that meet these criteria. We have introduced knockout alleles for the corresponding genes into the tobacco plastid genome. Five of the targeted genes (ribosomal protein of the large subunit22 [rpl22], rpl23, rpl32, ribosomal protein of the small subunit3 [rps3], and rps16) were shown to be essential even under heterotrophic conditions, despite their loss in at least some parasitic plastid-bearing lineages. This suggests that nonphotosynthetic plastids show elevated rates of gene transfer to the nuclear genome. Knockout of two ribosomal protein genes, rps15 and rpl36, yielded homoplasmic transplastomic mutants, thus indicating nonessentiality. Whereas Δrps15 plants showed only a mild phenotype, Δrpl36 plants were severely impaired in photosynthesis and growth and, moreover, displayed greatly altered leaf morphology. This finding provides strong genetic evidence that chloroplast translational activity influences leaf development, presumably via a retrograde signaling pathway.     

64 Nucleus-encoded, plastid-targeted glyceraldehyde-3-phosphate dehydrogenase (GAPDH) indicates a single origin for chromalveolate plastids

Plastids (the photosynthetic organelles of plants and algae) ultimately originated through an endosymbiosis between a cyanobacterium and a eukaryote. Subsequently, plastids spread to other eukaryotes by secondary endosymbioses that took place between a eukaryotic alga and a second eukaryote. Recently, evidence has mounted in favour of a single origin for plastids of apicomplexans, cryptophytes, dinoflagellates, haptophytes, and heterokonts (together with their non-photosynthetic relatives, collectively termed chromalveolates). As of yet, however, no single molecular marker has been described which supports a common origin for all of these plastids. One piece of the evidence for a single origin of chromalveolate plastids came from plastid-targeted glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which originated by a gene duplication of the cytosolic form. However, no plastid GAPDH has been characterized from haptophytes, leaving an important piece of the puzzle missing. We have sequenced genes encoding cytosolic, mitochondrial-targeted, and plastid-targeted GAPDH proteins from a number of haptophytes and heterokonts, and found the haptophyte homologues to branch within the strongly supported clade of chromalveolate plastid-targeted GAPDH genes. Interestingly, plastid-targeted GAPDH genes from the haptophytes were more closely related to apicomplexan genes than was expected. Overall, the evolution of plastid-targeted GAPDH reinforces other data for a red algal ancestry of apicomplexan plastids, and raises a number of questions about the importance of plastid loss and the possibility of cryptic plastids in non-photosynthetic lineages such as ciliates.     

Ancient recruitment by chromists of green algal genes encoding enzymes for carotenoid biosynthesis

Chromist algae (stramenopiles, cryptophytes, and haptophytes) are major contributors to marine primary productivity. These eukaryotes acquired their plastid via secondary endosymbiosis, whereby an early-diverging red alga was engulfed by a protist and the plastid was retained and its associated nuclear-encoded genes were transferred to the host genome. Current data suggest, however, that chromists are paraphyletic; therefore, it remains unclear whether their plastids trace back to a single secondary endosymbiosis or, alternatively, this organelle has resulted from multiple independent events in the different chromist lineages. Both scenarios, however, predict that plastid-targeted, nucleus-encoded chromist proteins should be most closely related to their red algal homologs. Here we analyzed the biosynthetic pathway of carotenoids that are essential components of all photosynthetic eukaryotes and find a mosaic evolutionary origin of these enzymes in chromists. Surprisingly, about one-third (5/16) of the proteins are most closely related to green algal homologs with three branching within or sister to the early-diverging Prasinophyceae. This phylogenetic association is corroborated by shared diagnostic indels and the syntenic arrangement of a specific gene pair involved in the photoprotective xanthophyll cycle. The combined data suggest that the prasinophyte genes may have been acquired before the ancient split of stramenopiles, haptophytes, cryptophytes, and putatively also dinoflagellates. The latter point is supported by the observed monophyly of alveolates and stramenopiles in most molecular trees. One possible explanation for our results is that the green genes are remnants of a cryptic endosymbiosis that occurred early in chromalveolate evolution; that is, prior to the postulated split of stramenopiles, alveolates, haptophytes, and cryptophytes. The subsequent red algal capture would have led to the loss or replacement of most green genes via intracellular gene transfer from the new endosymbiont. We argue that the prasinophyte genes were retained because they enhance photosynthetic performance in chromalveolates, thus extending the niches available to these organisms. The alternate explanation of green gene origin via serial endosymbiotic or horizontal gene transfers is also plausible, but the latter would require the independent origins of the same five genes in some or all the different chromalveolate lineages.     

Symbiotic origin of a novel actin gene in the cryptophyte Pyrenomonas helgolandii

Cryptophytes are photosynthetic protists that have acquired their plastids through the secondary symbiotic uptake of a red alga. A remarkable feature of cryptophytes is that they maintain a reduced form of the red algal nucleus, the nucleomorph, between the second and third plastid membranes (periplastidial compartment; PC). The nucleomorph is thought to be a transition state in the evolution of secondary plastids, with this genome ultimately being lost when photosynthesis comes under full control of the "host" nucleus (e.g., as in heterokonts, haptophytes, and euglenophytes). Genes presently found in the nucleomorph seem to be restricted to those involved in its own maintenance and to that of the plastid; other genes were lost as the endosymbiont was progressively reduced to its present state. Surprisingly, we found that the cryptophyte Pyrenomonas helgolandii possesses a novel type of actin gene that originated from the nucleomorph genome of the symbiont. Our results demonstrate for the first time that secondary symbionts can contribute genes to the host lineage which are unrelated to plastid function. These genes are akin to the products of gene duplication or lateral transfer and provide a source of evolutionary novelty that can significantly increase the genetic diversity of the host lineage. We postulate that this may be a common phenomenon in algae containing secondary plastids that has yet to be fully appreciated due to a dearth of evolutionary studies of nuclear genes in these taxa.     

Nuclear genome sequence of the plastid-lacking cryptomonad <Emphasis Type="Italic">Goniomonas avonlea</Emphasis> provides insights into the evolution of secondary plastids

Background

The evolution of photosynthesis has been a major driver in eukaryotic diversification. Eukaryotes have acquired plastids (chloroplasts) either directly via the engulfment and integration of a photosynthetic cyanobacterium (primary endosymbiosis) or indirectly by engulfing a photosynthetic eukaryote (secondary or tertiary endosymbiosis). The timing and frequency of secondary endosymbiosis during eukaryotic evolution is currently unclear but may be resolved in part by studying cryptomonads, a group of single-celled eukaryotes comprised of both photosynthetic and non-photosynthetic species. While cryptomonads such as Guillardia theta harbor a red algal-derived plastid of secondary endosymbiotic origin, members of the sister group Goniomonadea lack plastids. Here, we present the genome of Goniomonas avonlea—the first for any goniomonad—to address whether Goniomonadea are ancestrally non-photosynthetic or whether they lost a plastid secondarily.

Results

We sequenced the nuclear and mitochondrial genomes of Goniomonas avonlea and carried out a comparative analysis of Go. avonlea, Gu. theta, and other cryptomonads. The Go. avonlea genome assembly is ~?92 Mbp in size, with 33,470 predicted protein-coding genes. Interestingly, some metabolic pathways (e.g., fatty acid biosynthesis) predicted to occur in the plastid and periplastidal compartment of Gu. theta appear to operate in the cytoplasm of Go. avonlea, suggesting that metabolic redundancies were generated during the course of secondary plastid integration. Other cytosolic pathways found in Go. avonlea are not found in Gu. theta, suggesting secondary loss in Gu. theta and other plastid-bearing cryptomonads. Phylogenetic analyses revealed no evidence for algal endosymbiont-derived genes in the Go. avonlea genome. Phylogenomic analyses point to a specific relationship between Cryptista (to which cryptomonads belong) and Archaeplastida.

Conclusion

We found no convincing genomic or phylogenomic evidence that Go. avonlea evolved from a secondary red algal plastid-bearing ancestor, consistent with goniomonads being ancestrally non-photosynthetic eukaryotes. The Go. avonlea genome sheds light on the physiology of heterotrophic cryptomonads and serves as an important reference point for studying the metabolic “rewiring” that took place during secondary plastid integration in the ancestor of modern-day Cryptophyceae.

     

Diversity and evolutionary history of plastids and their hosts

By synthesizing data from individual gene phylogenies, large concatenated gene trees, and other kinds of molecular, morphological, and biochemical markers, we begin to see the broad outlines of a global phylogenetic tree of eukaryotes. This tree is apparently composed of five large assemblages, or "supergroups." Plants and algae, or more generally eukaryotes with plastids (the photosynthetic organelle of plants and algae and their nonphotosynthetic derivatives) are scattered among four of the five supergroups. This is because plastids have had a complex evolutionary history involving several endosymbiotic events that have led to their transmission from one group to another. Here, the history of the plastid and of its various hosts is reviewed with particular attention to the number and nature of the endosymbiotic events that led to the current distribution of plastids. There is accumulating evidence to support a single primary origin of plastids from a cyanobacterium (with one intriguing possible exception in the little-studied amoeba Paulinella), followed by the diversification of glaucophytes, red and green algae, with plants evolving from green algae. Following this, some of these algae were themselves involved in secondary endosymbiotic events. The best current evidence indicates that two independent secondary endosymbioses involving green algae gave rise to euglenids and chlorarachniophytes, whereas a single endosymbiosis with a red algae gave rise to the chromalveolates, a diverse group including cryptomonads, haptophytes, heterokonts, and alveolates. Dinoflagellates (alveolates) have since taken up other algae in serial secondary and tertiary endosymbioses, raising a number of controversies over the origin of their plastids, and by extension, the recently discovered cryptic plastid of the closely related apicomplexan parasites.     

SYMBIOTIC ORIGIN OF A NOVEL ACTIN GENE IN THE CRYPTOPHYTE,PYRENOMONAS HELGOLANDII

Cryptophytes are photosynthetic protists that have acquired their plastids through the secondary symbiotic uptake of a red alga. A remarkable feature of cryptophytes is that they maintain a reduced form of the red algal nucleus, the nucleomorph, between the second and third plastid membranes (periplastidial compartment, PC). The nucleomorph is thought to be a transition state in the evolution of secondary plastids with this genome ultimately being lost (e.g., as in heterokonts, haptophytes, euglenophytes) when photosynthesis comes under full control of the “host” nucleus. For this to happen, all genes for plastid function must be transferred from the nucleomorph to the nucleus. In this regard, it is generally assumed that nucleomorph genes with functions unrelated to plastid or PC maintenance are lost. Surprisingly, we show here the existence of a novel type of actin gene in the host nucleus of the cryptophyte, Pyrenomonas helgolandii, that has originated from the nucleomorph genome of the symbiont. Our results demonstrate for the first time that secondary symbionts can contribute genes to the host lineage that are unrelated to plastid function. These genes are akin to the products of gene duplication and provide a source of evolutionary novelty that could significantly increase the genetic diversity of the host lineage. We postulate that this may be a common phenomenon in algae containing secondary plastids that has yet to be fully appreciated due to a dearth of evolutionary studies of nuclear genes in these taxa.     

A cyanobacterial gene in nonphotosynthetic protists--an early chloroplast acquisition in eukaryotes?

Since the incorporation of mitochondria and chloroplasts (plastids) into the eukaryotic cell by endosymbiosis, genes have been transferred from the organellar genomes to the nucleus of the host, via an ongoing process known as endosymbiotic gene transfer. Accordingly, in photosynthetic eukaryotes, nuclear genes with cyanobacterial affinity are believed to have originated from endosymbiotic gene transfer from chloroplasts. Analysis of the Arabidopsis thaliana genome has shown that a significant fraction (2%-9%) of the nuclear genes have such an endosymbiotic origin. Recently, it was argued that 6-phosphogluconate dehydrogenase (gnd)-the second enzyme in the oxidative pentose phosphate pathway-was one such example. Here we show that gnd genes with cyanobacterial affinity also are present in several nonphotosynthetic protistan lineages, such as Heterolobosea, Apicomplexa, and parasitic Heterokonta. Current data cannot definitively resolve whether these groups acquired the gnd gene by primary and/or secondary endosymbiosis or via an independent lateral gene transfer event. Nevertheless, our data suggest that chloroplasts were introduced into eukaryotes much earlier than previously thought and that several major groups of heterotrophic eukaryotes have secondarily lost photosynthetic plastids.     

THE EVOLUTION OF RNA POLYMERASE II IN RED ALGAE

Cryptophytes are photosynthetic protists that have acquired their plastids through the secondary symbiotic uptake of a red alga. A remarkable feature of cryptophytes is that they maintain a reduced form of the red algal nucleus, the nucleomorph, between the second and third plastid membranes (periplastidial compartment, PC). The nucleomorph is thought to be a transition state in the evolution of secondary plastids with this genome ultimately being lost (e.g., as in heterokonts, haptophytes, euglenophytes) when photosynthesis comes under full control of the "host" nucleus. For this to happen, all genes for plastid function must be transferred from the nucleomorph to the nucleus. In this regard, it is generally assumed that nucleomorph genes with functions unrelated to plastid or PC maintenance are lost. Surprisingly, we show here the existence of a novel type of actin gene in the host nucleus of the cryptophyte, Pyrenomonas helgolandii , that has originated from the nucleomorph genome of the symbiont. Our results demonstrate for the first time that secondary symbionts can contribute genes to the host lineage that are unrelated to plastid function. These genes are akin to the products of gene duplication and provide a source of evolutionary novelty that could significantly increase the genetic diversity of the host lineage. We postulate that this may be a common phenomenon in algae containing secondary plastids that has yet to be fully appreciated due to a dearth of evolutionary studies of nuclear genes in these taxa.