The Case for a Single‐Plastid Origin Revisited: Convergent Evolution in Organellar Gene Content

Three eukaryotic lineages generally are believed to have plastids that are primary in origin; that is, descended directly from a cyanobacterial endosymbiont. The recovery of these plastids as a monophyletic group in most molecular phylogenetic analyses, along with similarities in genome content and protein targeting mechanisms, have been cited as strong evidence in support of the hypothesis of a single endosymbiotic origin of all plastids. Although these data indeed are consistent with a single plastid origin, they also are consistent with the proposition of multiple endosymbiotic origins. Each hypothesis requires certain evolutionary assumptions in order to be reconciled with all existing data; at present, it is unclear which of these assumptions most likely reflect the historical process that gave rise to plastid diversity. Here we examine similarities in gene content among representatives of the three primary plastid lineages, using as a control the genome of a mitochondrion that almost certainly originated as an independent endosymbiotic association. To minimize metabolic constraints on gene retention we focus on two datasets, ribosomal protein and transfer RNA genes, neither of which is tied directly to specific organellar functions. Analyses of all possible pair‐wise comparisons among the three plastids and mitochondrion indicate that genomic similarities are most consistent with convergent evolution due to constraints on gene loss, rather than with hypothesized shared evolutionary histories. We find no evidence of phylogenetic signal in the pattern of gene loss overlying this convergence. In light of these results, we address other lines of evidence and arguments that have been raised in support of a single plastid origin.     

THE SYMBIOTIC BIRTH AND SPREAD OF PLASTIDS: HOW MANY TIMES AND WHODUNIT?

I discuss the evidence for a single origin of primary plastids in the context of a paper in this issue challenging this view, and I review recent evidence concerning the number of secondary plastid endosymbioses and the controversy over whether the relic plastid of apicomplexans is of red or green algal origin. A broad consensus has developed that the plastids of green algae, red algae, and glaucophytes arose from the same primary, cyanobacterial endosymbiosis. Although the analyses in this issue by Stiller and colleagues firmly undermine one of many sources of data, gene content similarities among plastid genomes used to argue for a monophyletic origin of primary plastids, the overall evidence still clearly favors monophyly. Nonetheless, this issue should not be considered settled and new data should be sought from better sampling of cyanobacteria and glaucophytes, from sequenced nuclear genomes, and from careful analysis of such key features as the plastid import apparatus. With respect to the number of secondary plastid symbioses, it is completely unclear as to whether the secondary plastids of euglenophytes and chlorarachniophytes arose by the same or two different algal endosymbioses. Recent analyses of certain plastid and nuclear genes support the chromalveolate hypothesis of Cavalier-Smith, namely, that the plastids of heterokonts, haptophytes, cryptophytes, dinoflagellates, and apicomplexans all arose from a common endosymbiosis involving a red alga. However, another recent paper presents intriguing conflicting data on this score for one of these groups—apicomplexans—arguing instead that they acquired their plastids from green algae.     

Chromalveolates and the Evolution of Plastids by Secondary Endosymbiosis1

ABSTRACT. The establishment of a new plastid organelle by secondary endosymbiosis represents a series of events of massive complexity, and yet we know it has taken place multiple times because both green and red algae have been taken up by other eukaryotic lineages. Exactly how many times these events have succeeded, however, has been a matter of debate that significantly impacts how we view plastid evolution, protein targeting, and eukaryotic relationships. On the green side it is now largely accepted that two independent events led to plastids of euglenids and chlorarachniophytes. How many times red algae have been taken up is less clear, because there are many more lineages with red alga‐derived plastids (cryptomonads, haptophytes, heterokonts, dinoflagellates and apicomplexa) and the relationships between these lineages are less clear. Ten years ago, Cavalier‐Smith proposed that these plastids were all derived from a single endosymbiosis, an idea that was dubbed the chromalveolate hypothesis. No one observation has yet supported the chromalveolate hypothesis as a whole, but molecular data from plastid‐encoded and plastid‐targeted proteins have provided strong support for several components of the overall hypothesis, and evidence for cryptic plastids and new photosynthetic lineages (e.g. Chromera) have transformed our view of plastid distribution within the group. Collectively, these data are most easily reconciled with a single origin of the chromalveolate plastids, although the phylogeny of chromalveolate host lineages (and potentially Rhizaria) remain to be reconciled with this plastid data.     

Origins of plastids and glyceraldehyde-3-phosphate dehydrogenase genes in the green-colored dinoflagellate Lepidodinium chlorophorum

The dinoflagellate Lepidodinium chlorophorum possesses "green" plastids containing chlorophylls a and b (Chl a+b), unlike most dinoflagellate plastids with Chl a+c plus a carotenoid peridinin (peridinin-containing plastids). In the present study we determined 8 plastid-encoded genes from Lepidodinium to investigate the origin of the Chl a+b-containing dinoflagellate plastids. The plastid-encoded gene phylogeny clearly showed that Lepidodinium plastids were derived from a member of Chlorophyta, consistent with pigment composition. We also isolated three different glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes from Lepidodinium-one encoding the putative cytosolic "GapC" enzyme and the remaining two showing affinities to the "plastid-targeted GapC" genes. In a GAPDH phylogeny, one of the plastid-targeted GapC-like sequences robustly grouped with those of dinoflagellates bearing peridinin-containing plastids, while the other was nested in a clade of the homologues of haptophytes and dinoflagellate genera Karenia and Karlodinium bearing "haptophyte-derived" plastids. Since neither host nor plastid phylogeny suggested an evolutionary connection between Lepidodinium and Karenia/Karlodinium, a lateral transfer of a plastid-targeted GapC gene most likely took place from a haptophyte or a dinoflagellate with haptophyte-derived plastids to Lepidodinium. The plastid-targeted GapC data can be considered as an evidence for the single origin of plastids in haptophytes, cryptophytes, stramenopiles, and alveolates. However, in the light of Lepidodinium GAPDH data, we need to closely examine whether the monophyly of the plastids in the above lineages inferred from plastid-targeted GapC genes truly reflects that of the host lineages.     

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.     

Babesia bovis: A comprehensive phylogenetic analysis of plastid-encoded genes supports green algal origin of apicoplasts

Apicomplexan parasites commonly contain a unique, non-photosynthetic plastid-like organelle termed the apicoplast. Previous analyses of other plastid-containing organisms suggest that apicoplasts were derived from a red algal ancestor. In this report, we present an extensive phylogenetic study of apicoplast origins using multiple previously reported apicoplast sequences as well as several sequences recently reported. Phylogenetic analysis of amino acid sequences was used to determine the evolutionary origin of the organelle. A total of nine plastid genes from 37 species were incorporated in our study. The data strongly support a green algal origin for apicoplasts and Euglenozoan plastids. Further, the nearest green algae lineage to the Apicomplexans is the parasite Helicosporidium, suggesting that apicoplasts may have originated by lateral transfer from green algal parasite lineages. The results also substantiate earlier findings that plastids found in Heterokonts such as Odontella and Thalassiosira were derived from a separate secondary endosymbiotic event likely originating from a red algal lineage.     

Gene phylogenies and the endosymbiotic origin of plastids.

The endosymbiotic origin of chloroplasts from cyanobacteria has long been suspected and has been confirmed in recent years by many lines of evidence. Debate now is centered on whether plastids are derived from a single endosymbiotic event or from multiple events involving several photosynthetic prokaryotes and/or eukaryotes. Phylogenetic analysis was undertaken using the inferred amino acid sequences from the genes psbA, rbcL, rbcS, tufA and atpB and a published analysis (Douglas and Turner, 1991) of nucleotide sequences of small subunit (SSU) rRNA to examine the relationships among purple bacteria, cyanobacteria and the plastids of non-green algae (including rhodophytes, chromophytes, a cryptophyte and a glaucophyte), green algae, euglenoids and land plants. Relationships within and among groups are generally consistent among all the trees; for example, prochlorophytes cluster with cyanobacteria (and not with green plastids) in each of the trees and rhodophytes are ancestral to or the sister group of the chromophyte algae. One notable exception is that Euglenophytes are associated with the green plastid lineage in psbA, rbcL, rbcS and tufA trees and with the non-green plastid lineage in SSU rRNA trees. Analysis of psbA, tufA, atpB and SSU rRNA sequences suggests that only a single bacterial endosympbiotic event occurred leading to plastids in the various algal and plant lineages. In contrast, analysis of rbcL and rbcS sequences strongly suggests that plastids are polyphyletic in origin, with plastids being derived independently from both purple bacteria and cyanobacteria. A hypothesis consistent with these discordant trees is that a single bacterial endosymbiotic event occurred leading to all plastids, followed by the lateral transfer of the rbcLS operon from a purple bacterium to a rhodophyte.     

Identification of two origins of replication in the single chromosome of the archaeon Sulfolobus solfataricus

Eukaryotic chromosomes possess multiple origins of replication, whereas bacterial chromosomes are replicated from a single origin. The archaeon Pyrococcus abyssi also appears to have a single origin, suggesting a common rule for prokaryotes. However, in the current work, we describe the identification of two active origins of replication in the single chromosome of the hyperthermophilic archaeon Sulfolobus solfataricus. Further, we identify conserved sequence motifs within the origins that are recognized by a family of three Sulfolobus proteins that are homologous to the eukaryotic initiator proteins Orc1 and Cdc6. We demonstrate that the two origins are recognized by distinct subsets of these Orc1/Cdc6 homologs. These data, in conjunction with an analysis of the levels of the three Orc1/Cdc6 proteins in different growth phases and cell cycle stages, lead us to propose a model for the roles for these proteins in modulating origin activity.     

Evolution: red algal genome affirms a common origin of all plastids

Photosynthetic organelles (plastids) come in many forms and were originally thought to have multiple origins. The complete genome of the thermophilic red alga Cyanidioschizon merolae provides further evidence that all plastids derive from a single endosymbiotic event more than 600 million years ago.     

Heterotachy processes in rhodophyte-derived secondhand plastid genes: Implications for addressing the origin and evolution of dinoflagellate plastids

Serial transfer of plastids from one eukaryotic host to another is the key process involved in evolution of secondhand plastids. Such transfers drastically change the environment of the plastids and hence the selection regimes, presumably leading to changes over time in the characteristics of plastid gene evolution and to misleading phylogenetic inferences. About half of the dinoflagellate protists species are photosynthetic and unique in harboring a diversity of plastids acquired from a wide range of eukaryotic algae. They are therefore ideal for studying evolutionary processes of plastids gained through secondary and tertiary endosymbioses. In the light of these processes, we have evaluated the origin of 2 types of dinoflagellate plastids, containing the peridinin or 19'-hexanoyloxyfucoxanthin (19'-HNOF) pigments, by inferring the phylogeny using "covarion" evolutionary models allowing the pattern of among-site rate variation to change over time. Our investigations of genes from secondary and tertiary plastids derived from the rhodophyte plastid lineage clearly reveal "heterotachy" processes characterized as stationary covarion substitution patterns and changes in proportion of variable sites across sequences. Failure to accommodate covarion-like substitution patterns can have strong effects on the plastid tree topology. Importantly, multigene analyses performed with probabilistic methods using among-site rate and covarion models of evolution conflict with proposed single origin of the peridinin- and 19'-HNOF-containing plastids, suggesting that analysis of secondhand plastids can be hampered by convergence in the evolutionary signature of the plastid DNA sequences. Another type of sequence convergence was detected at protein level involving the psaA gene. Excluding the psaA sequence from a concatenated protein alignment grouped the peridinin plastid with haptophytes, congruent with all DNA trees. Altogether, taking account of complex processes involved in the evolution of dinoflagellate plastid sequences (both at the DNA and amino acid level), we demonstrate the difficulty of excluding independent, tertiary origin for both the peridinin and 19'-HNOF plastids involving engulfment of haptophyte-like algae. In addition, the refined topologies suggest the red algal order, Porphyridales, as the endosymbiont ancestor of the secondary plastids in cryptophytes, haptophytes, and heterokonts.     

Did some red alga‐derived plastids evolve via kleptoplastidy? A hypothesis

The evolution of plastids has a complex and still unresolved history. These organelles originated from a cyanobacterium via primary endosymbiosis, resulting in three eukaryotic lineages: glaucophytes, red algae, and green plants. The red and green algal plastids then spread via eukaryote–eukaryote endosymbioses, known as secondary and tertiary symbioses, to numerous heterotrophic protist lineages. The number of these horizontal plastid transfers, especially in the case of red alga‐derived plastids, remains controversial. Some authors argue that the number of plastid origins should be minimal due to perceived difficulties in the transformation of a eukaryotic algal endosymbiont into a multimembrane plastid, but increasingly the available data contradict this argument. I suggest that obstacles in solving this dilemma result from the acceptance of a single evolutionary scenario for the endosymbiont‐to‐plastid transformation formulated by Cavalier‐Smith & Lee (1985). Herein I discuss data that challenge this evolutionary scenario. Moreover, I propose a new model for the origin of multimembrane plastids belonging to the red lineage and apply it to the dinoflagellate peridinin plastid. The new model has several general and practical implications, such as the requirement for a new definition of cell organelles and in the construction of chimeric organisms.     

Phylogeny of the Bangiophycidae (Rhodophyta) and the secondary endosymbiotic origin of algal plastids

The Rhodophyta (red algae) are composed of the subclasses Bangiophycidae and Florideophycidae. Two evolutionarily interesting features of the Bangiophycidae are: (1) they are the ancestral pool from which the more morphologically complex taxa in the Florideophycidae have arisen and (2) they are the sources of the plastids, through secondary endosymbioses, for the Cryptophyta, Haptophyta, and the Heterokonta. To understand Bangiophycidae phylogeny and to gain further insights into red algal secondary endosymbioses, we sequenced the plastid-encoded small subunit ribosomal DNA (rDNA) coding region from nine members of this subclass and from two members of the Florideophycidae. These sequences were included in phylogenetic analyses with all available red algal plus chlorophyll a + c algal plastid rDNA coding regions. Our results are consistent with a monophyletic origin of the Florideophycidae with these taxa forming a sister group of the Bangiales. The Bangiophycidae is of a paraphyletic origin with orders such as the Porphyridiales polyphyletic and distributed over three independent red algal lineages. The plastids of the heterokonts are most closely related to members of the Cyanidium-Galdieria group of Porphyridiales and are not directly related to cryptophyte and haptophyte plastids. The phylogenies provide strong evidence for the independent origins of these "complex" algal plastids from different members of the Bangiophycidae.     

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.     

WEIGHING THE EVIDENCE FOR A SINGLE ORIGIN OF PLASTIDS1

The Journal of Phycology recently published a research paper entitled “A single origin of plastids revisited: convergent evolution in organellar genome content” ( Stiller et al. 2003 ). Also appearing in that issue was a minireview by Jeffrey D. Palmer ( Palmer 2003 ), “The symbiotic birth and spread of plastids: how many times and whodunit?” In his review, Palmer discussed evidence in support of a single endosymbiotic origin, in light of our analyses showing that similarities in plastid genome content are explained better by convergent evolution than by shared evolutionary history. Palmer raised a number of important issues that were not addressed in our paper, including the point that, in his view, no real evidence has been cited against a single plastid origin. After carefully considering Palmer's arguments, and this key point in particular, I am prompted to offer a few additional comments in the spirit of furthering a useful discussion begun in the February issue.     

Two compound replication origins in Saccharomyces cerevisiae contain redundant origin recognition complex binding sites

While many of the proteins involved in the initiation of DNA replication are conserved between yeasts and metazoans, the structure of the replication origins themselves has appeared to be different. As typified by ARS1, replication origins in Saccharomyces cerevisiae are <150 bp long and have a simple modular structure, consisting of a single binding site for the origin recognition complex, the replication initiator protein, and one or more accessory sequences. DNA replication initiates from a discrete site. While the important sequences are currently less well defined, metazoan origins appear to be different. These origins are large and appear to be composed of multiple, redundant elements, and replication initiates throughout zones as large as 55 kb. In this report, we characterize two S. cerevisiae replication origins, ARS101 and ARS310, which differ from the paradigm. These origins contain multiple, redundant binding sites for the origin recognition complex. Each binding site must be altered to abolish origin function, while the alteration of a single binding site is sufficient to inactivate ARS1. This redundant structure may be similar to that seen in metazoan origins.     

Supertrees disentangle the chimerical origin of eukaryotic genomes

Eukaryotes are traditionally considered to be one of the three natural divisions of the tree of life and the sister group of the Archaebacteria. However, eukaryotic genomes are replete with genes of eubacterial ancestry, and more than 20 mutually incompatible hypotheses have been proposed to account for eukaryote origins. Here we test the predictions of these hypotheses using a novel supertree-based phylogenetic signal-stripping method, and recover supertrees of life based on phylogenies for up to 5,741 single gene families distributed across 185 genomes. Using our signal-stripping method, we show that there are three distinct phylogenetic signals in eukaryotic genomes. In order of strength, these link eukaryotes with the Cyanobacteria, the Proteobacteria, and the Thermoplasmatales, an archaebacterial (euryarchaeotes) group. These signals correspond to distinct symbiotic partners involved in eukaryote evolution: plastids, mitochondria, and the elusive host lineage. According to our whole-genome data, eukaryotes are hardly the sister group of the Archaebacteria, because up to 83% of eukaryotic genes with a prokaryotic homolog have eubacterial, not archaebacterial, origins. The results reject all but two of the current hypotheses for the origin of eukaryotes: those assuming a sulfur-dependent or hydrogen-dependent syntrophy for the origin of mitochondria.     

Molecular phylogenies of plastid origins and algal evolution

Summary An overview of recent molecular analyses regarding origins of plastids in algal lineages is presented. Since different phylogenetic analyses can yield contradictory views of algal plastid origins, we have examined the effect of two distance measurement methods and two distance matrix tree-building methods upon topologies for the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit nucleotide sequence data set. These results are contrasted to those from bootstrap parsimony analysis of nucleotide sequence data subsets. It is shown that the phylogenetic information contained within nucleotide sequences for the chloroplast-encoded gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, integral to photosynthesis, indicates an independent origin for this plastid gene in different plant taxa. This finding is contrasted to contrary results derived from 16S rRNA sequences. Possible explanations for discrepancies observed for these two different molecules are put forth. Other molecular sequence data which address questions of early plant evolution and the eubacterial origins of algal organelles are discussed. Offprint requests to: W. Martin     

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.     

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.