Photosynthetic eukaryotes unite: endosymbiosis connects the dots

The photosynthetic organelle of algae and plants (the plastid) traces its origin to a primary endosymbiotic event in which a previously non-photosynthetic protist engulfed and enslaved a cyanobacterium. This eukaryote then gave rise to the red, green and glaucophyte algae. However, many algal lineages, such as the chlorophyll c-containing chromists, have a more complicated evolutionary history involving a secondary endosymbiotic event, in which a protist engulfed an existing eukaryotic alga (in this case, a red alga). Chromists such as diatoms and kelps then rose to great importance in aquatic habitats. Another algal group, the dinoflagellates, has undergone tertiary (engulfment of a secondary plastid) and even quaternary endosymbioses. In this review, we examine algal diversity and show endosymbiosis to be a major force in algal evolution. This area of research has advanced rapidly and long-standing issues such as the chromalveolate hypothesis and the extent of endosymbiotic gene transfer have recently been clarified.     

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.     

Multiple genes of apparent algal origin suggest ciliates may once have been photosynthetic

Plantae (as defined by Cavalier-Smith, 1981) plastids evolved via primary endosymbiosis whereby a heterotrophic protist enslaved a photosynthetic cyanobacterium. This "primary" plastid spread into other eukaryotes via secondary endosymbiosis. An important but contentious theory in algal evolution is the chromalveolate hypothesis that posits chromists (cryptophytes, haptophytes, and stramenopiles) and alveolates (ciliates, apicomplexans, and dinoflagellates) share a common ancestor that contained a red-algal-derived "secondary" plastid. Under this view, the existence of several later-diverging plastid-lacking chromalveolates such as ciliates and oomycetes would be explained by plastid loss in these lineages. To test the idea of a photosynthetic ancestry for ciliates, we used the 27,446 predicted proteins from the macronuclear genome of Tetrahymena thermophila to query prokaryotic and eukaryotic genomes. We identified 16 proteins of possible algal origin in the ciliates Tetrahymena and Paramecium tetraurelia. Fourteen of these are present in other chromalveolates. Here we compare and contrast the likely scenarios for algal-gene origin in ciliates either via multiple rounds of horizontal gene transfer (HGT) from algal prey or symbionts, or through endosymbiotic gene transfer (EGT) during a putative photosynthetic phase in their evolution.     

Glutamate synthase is plastid-encoded in a red alga: implications for the evolution of glutamate synthases

An actively transcribed gene (glsF) encoding for ferredoxin-dependent glutamate synthase (Fd-GOGAT) was found on the plastid genome of the multicellular red alga Antithamnion sp. Fd-GOGAT is not plastid-encoded in chlorophytic plants, demonstrating that red algal plastid genomes encode for additional functions when compared to those known from green chloroplasts. Moreover, our results suggest that the plant Fd-GOGAT has an endosymbiotic origin. The same may not be true for NADPH-dependent GOGAT. In Antithamnion glsF is flanked upstream by cpcBA and downstream by psaC and is transcribed monocistronically. Implications of these results for the evolution of GOGAT enzymes and the plastid genome are discussed.     

A SINGLE ORIGIN OF PLASTIDS REVISITED: CONVERGENT EVOLUTION IN ORGANELLAR GENOME CONTENT1

In recent years a consensus has emerged from molecular phylogenetic investigations favoring a common endosymbiotic ancestor for all chloroplasts. It is within this conceptual framework that most comparative analyses of eukaryotic biochemistry and genetics now are interpreted. One of the first and most influential sources of data leading to this consensus is the remarkable similarity in genome content among all major plastid lineages. Here we report statistical analyses of two sequence data sets, genes encoding ribosomal proteins and transfer RNAs, from representatives of the three primary plastid lineages and a mitochondrion. The latter almost certainly originated in an independent endosymbiotic association and serves as a control for similarity due to convergent evolution. When genes related to organelle‐specific function are factored out, plastid genomes appear to be no more similar to each other than they are to the mitochondrion. Total similarities in gene content, measured as deviations from the expectation from a process of random gene loss, are correlated with the extent of reduction in the two genomes compared. They do not appear to reflect putative evolutionary relationships among plastids. These analyses indicate that similarities in plastid genome content are better explained by convergent evolution due to constraint on gene loss than by a shared evolutionary history. A review of other data cited as support for a single plastid origin suggests that the alternative hypothesis of multiple origins is at least equally consistent in most cases.     

Plastid evolution: origins, diversity, trends

The amazing diversity of extant photosynthetic eukaryotes is largely a result of the presence of formerly free-living photosynthesizing organisms that have been sequestered by eukaryotic hosts and established as plastids in a process known as endosymbiosis. The evolutionary history of these endosymbiotic events was traditionally investigated by studying ultrastructural features and pigment characteristics but in recent years has been approached using molecular sequence data and gene trees. Two important developments, more detailed studies of members of the Cyanobacteria (from which plastids ultimately derive) and the availability of complete plastid genome sequences from a wide variety of plant and algal lineages, have allowed a more accurate reconstruction of plastid evolution.     

Nucleomorph genomes: structure, function, origin and evolution

The cryptomonads and chlorarachniophytes are two unicellular algal lineages with complex cellular structures and fascinating evolutionary histories. Both groups acquired their photosynthetic abilities through the assimilation of eukaryotic endosymbionts. As a result, they possess two distinct cytosolic compartments and four genomes--two nuclear genomes, an endosymbiont-derived plastid genome and a mitochondrial genome derived from the host cell. Like mitochondrial and plastid genomes, the genome of the endosymbiont nucleus, or 'nucleomorph', of cryptomonad and chlorarachniophyte cells has been greatly reduced through the combined effects of gene loss and intracellular gene transfer. This article focuses on the structure, function, origin and evolution of cryptomonad and chlorarachniophyte nucleomorph genomes in light of recent comparisons of genome sequence data from both groups. It is now possible to speculate on the reasons that nucleomorphs persist in cryptomonads and chlorarachniophytes but have been lost in all other algae with plastids of secondary endosymbiotic origin.     

Chimeric plastid proteome in the Florida "red tide" dinoflagellate Karenia brevis

Current understanding of the plastid proteome comes almost exclusively from studies of plants and red algae. The proteome in these taxa has a relatively simple origin via integration of proteins from a single cyanobacterial primary endosymbiont and the host. However, the most successful algae in marine environments are the chlorophyll c-containing chromalveolates such as diatoms and dinoflagellates that contain a plastid of red algal origin derived via secondary or tertiary endosymbiosis. Virtually nothing is known about the plastid proteome in these taxa. We analyzed expressed sequence tag data from the toxic "Florida red tide" dinoflagellate Karenia brevis that has undergone a tertiary plastid endosymbiosis. Comparative analyses identified 30 nuclear-encoded plastid-targeted proteins in this chromalveolate that originated via endosymbiotic or horizontal gene transfer (HGT) from multiple different sources. We identify a fundamental divide between plant/red algal and chromalveolate plastid proteomes that reflects a history of mixotrophy in the latter group resulting in a highly chimeric proteome. Loss of phagocytosis in the "red" and "green" clades effectively froze their proteomes, whereas chromalveolate lineages retain the ability to engulf prey allowing them to continually recruit new, potentially adaptive genes through subsequent endosymbioses and HGT. One of these genes is an electron transfer protein (plastocyanin) of green algal origin in K. brevis that likely allows this species to thrive under conditions of iron depletion.     

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.     

Hypothesis: Gene‐rich plastid genomes in red algae may be an outcome of nuclear genome reduction

Red algae (Rhodophyta) putatively diverged from the eukaryote tree of life >1.2 billion years ago and are the source of plastids in the ecologically important diatoms, haptophytes, and dinoflagellates. In general, red algae contain the largest plastid gene inventory among all such organelles derived from primary, secondary, or additional rounds of endosymbiosis. In contrast, their nuclear gene inventory is reduced when compared to their putative sister lineage, the Viridiplantae, and other photosynthetic lineages. The latter is thought to have resulted from a phase of genome reduction that occurred in the stem lineage of Rhodophyta. A recent comparative analysis of a taxonomically broad collection of red algal and Viridiplantae plastid genomes demonstrates that the red algal ancestor encoded ~1.5× more plastid genes than Viridiplantae. This difference is primarily explained by more extensive endosymbiotic gene transfer (EGT) in the stem lineage of Viridiplantae, when compared to red algae. We postulate that limited EGT in Rhodophytes resulted from the countervailing force of ancient, and likely recurrent, nuclear genome reduction. In other words, the propensity for nuclear gene loss led to the retention of red algal plastid genes that would otherwise have undergone intracellular gene transfer to the nucleus. This hypothesis recognizes the primacy of nuclear genome evolution over that of plastids, which have no inherent control of their gene inventory and can change dramatically (e.g., secondarily non‐photosynthetic eukaryotes, dinoflagellates) in response to selection acting on the host lineage.     

More membranes, more proteins: complex protein import mechanisms into secondary plastids

Plastids are found across the tree of life in a tremendous diversity of life forms. Surprisingly they are not limited to photosynthetic organisms but also found in numerous predators and parasites. An important reason for the pervasiveness of plastids has been their ability to move laterally and to jump from one branch of the tree of life to the next through secondary endosymbiosis. Eukaryotic algae have entered endosymbiotic relationships with other eukaryotes on multiple independent occasions. The descendants of these endosymbiotic events now carry complex plastids, organelles that are bound by three or even four membranes. As in all endosymbiotic organelles most of the symbiont's genes have been transferred to the host and their protein products have to be imported into the organelle. As four membranes might suggest, this is a complex process. The emerging mechanisms display a series of translocons that mirror the divergent ancestry of the membranes they cross. This review is written from the viewpoint of a parasite biologist and seeks to provide a brief overview of plastid evolution in particular for readers not already familiar with plant and algal biology and then focuses on recent molecular discoveries using genetically tractable Apicomplexa and diatoms.     

The acquisition of phototrophy: adaptive strategies of hosting endosymbionts and organelles

Many non-photosynthetic species of protists and metazoans are capable of hosting viable algal endosymbionts or their organelles through adaptations of phagocytic pathways. A form of mixotrophy combining phototrophy and heterotrophy, acquired phototrophy (AcPh) encompasses a suite of endosymbiotic and organelle retention interactions, that range from facultative to obligate. AcPh is a common phenomenon in aquatic ecosystems, with endosymbiotic associations generally more prevalent in nutrient poor environments, and organelle retention typically associated with more productive ones. All AcPhs benefit from enhanced growth due to access to photosynthetic products; however, the degree of metabolic integration and dependency in the host varies widely. AcPh is found in at least four of the major eukaryotic supergroups, and is the driving force in the evolution of secondary and tertiary plastid acquisitions. Mutualistic resource partitioning characterizes most algal endosymbiotic interactions, while organelle retention is a form of predation, characterized by nutrient flow (i.e., growth) in one direction. AcPh involves adaptations to recognize specific prey or endosymbionts and to house organelles or endosymbionts within the endomembrane system but free from digestion. In many cases, hosts depend upon AcPh for the production of essential nutrients, many of which remain obscure. The practice of AcPh has led to multiple independent secondary and tertiary plastid acquisition events among several eukaryote lineages, giving rise to the diverse array of algae found in modern aquatic ecosystems. This article highlights those AcPhs that are model research organisms for both metazoans and protists. Much of the basic biology of AcPhs remains enigmatic, particularly (1) which essential nutrients or factors make certain forms of AcPh obligatory, (2) how hosts regulate and manipulate endosymbionts or sequestered organelles, and (3) what genomic imprint, if any, AcPh leaves on non-photosynthetic host species.     

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.     

When Less is More: Red Algae as Models for Studying Gene Loss and Genome Evolution in Eukaryotes

Genome evolution is usually viewed through the lens of growth in size and complexity over time, exemplified by plants and animals. In contrast, genome reduction is associated with a narrowing of ecological potential, such as in parasites and endosymbionts. But, can nuclear genome reduction also occur in, and potentially underpin a major radiation of free-living eukaryotes? An intriguing example of this phenomenon is provided by the red algae (Rhodophyta) that have lost many conserved pathways such as for flagellar motility, macroautophagy regulation, and phytochrome based light sensing. This anciently diverged, species-rich, and ecologically important algal lineage has undergone at least two rounds of large-scale genome reduction during its >1 billion-year evolutionary history. Here, using recent analyses of genome data, we review knowledge about the evolutionary trajectory of red algal nuclear and organelle gene inventories and plastid encoded autocatalytic introns. We compare and contrast Rhodophyta genome evolution to Viridiplantae (green algae and plants), both of which are members of the Archaeplastida, and highlight their divergent paths. We also discuss evidence for the speculative hypothesis that reduction in red algal plastid genome size through endosymbiotic gene transfer is counteracted by ongoing selection for compact nuclear genomes in red algae. Finally, we describe how the spliceosomal intron splicing apparatus provides an example of “evolution in action” in Rhodophyta and how the overall constraints on genome size in this lineage has left significant imprints on this key step in RNA maturation. Our review reveals the red algae to be an exciting, yet under-studied model that offers numerous novel insights as well as many unanswered questions that remain to be explored using modern genomic, genetic, and biochemical methods. The fact that a speciose lineage of free-living eukaryotes has spread throughout many aquatic habitats after having lost about 25% of its primordial gene inventory challenges us to elucidate the mechanisms underlying this remarkable feat.     

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.     

Dinoflagellate expressed sequence tag data indicate massive transfer of chloroplast genes to the nuclear genome

The peridinin-pigmented plastids of dinoflagellates are very poorly understood, in part because of the paucity of molecular data available from these endosymbiotic organelles. To identify additional gene sequences that would carry information about the biology of the peridinin-type dinoflagellate plastid and its evolutionary history, an analysis was undertaken of arbitrarily selected sequences from cDNA libraries constructed from Lingulodinium polyedrum (1012 non-redundant sequences) and Amphidinium carterae (2143). Among the two libraries 118 unique plastid-associated sequences were identified, including 30 (most from A. carterae) that are encoded in the plastid genome of the red alga Porphyra. These sequences probably represent bona fide nuclear genes, and suggest that there has been massive transfer of genes from the plastid to the nuclear genome in dinoflagellates. These data support the hypothesis that the peridinin-type plastid has a minimal genome, and provide data that contradict the hypothesis that there is an unidentified canonical genome in the peridinin-type plastid. Sequences were also identified that were probably transferred directly from the nuclear genome of the red algal endosymbiont, as well as others that are distinctive to the Alveolata. A preliminary report of these data was presented at the Botany 2002 meeting in Madison, WI.     

Cyanobacterial contribution to algal nuclear genomes is primarily limited to plastid functions

A single cyanobacterial primary endosymbiosis that occurred approximately 1.5 billion years ago is believed to have given rise to the plastid in the common ancestor of the Plantae or Archaeplastida--the eukaryotic supergroup comprising red, green (including land plants), and glaucophyte algae. Critical to plastid establishment was the transfer of endosymbiont genes to the host nucleus (i.e., endosymbiotic gene transfer [EGT]). It has been postulated that plastid-derived EGT played a significant role in plant nuclear-genome evolution, with 18% (or 4,500) of all nuclear genes in Arabidopsis thaliana having a cyanobacterial origin with about one-half of these recruited for nonplastid functions. Here, we determine whether the level of cyanobacterial gene recruitment proposed for Arabidopsis is of the same magnitude in the algal sisters of plants by analyzing expressed-sequence tag (EST) data from the glaucophyte alga Cyanophora paradoxa. Bioinformatic analysis of 3,576 Cyanophora nuclear genes shows that 10.8% of these with significant database hits are of cyanobacterial origin and one-ninth of these have nonplastid functions. Our data indicate that unlike plants, early-diverging algal groups appear to retain a smaller number of endosymbiont genes in their nucleus, with only a minor proportion of these recruited for nonplastid functions.     

Origin and ,evolution of organelle genomes

Molecular data (particularly sequence analyses) have established that two eukaryotic organelles, the mitochondrion and the plastid, are the descendants of endosymbiotic (eu)bacteria whose closest living relatives are the α-Proteobacteria (mitochondrion) and Cyanobacteria (plastid). This review describes recent data that favor the view that each organelle arose via this primary endosymbiotic pathway only once (monophyletic origin), such as the discovery of group I introns that appear to be structurally homologous and have identical insertion sites in metaphyte, chlorophyte and fungal mitochondrial genomes. However, it is also evident that the plastids in certain algal groups were acquired secondarily through a eukaryotic rather than a prokaryotic endosymbiont.     

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.