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.     

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.     

The Cyanelles of Cyanophora Paradoxa

The cyanelles of Cyanophora paradoxa, plastids surrounded by a peptidoglycan wall, are considered as a surviving example for an early stage of plastid evolution from endosymbiotic cyanobacteria. We highlight the model character of the system by focusing on three aspects: “organelle wall” structure, plastid genome organization, and protein translocation.

The biosynthetic pathway for cyanelle peptidoglycan appears to be analogous to that in Escherichia coli. Also, the basic structure of this peculiar organelle wall corresponds to that of the E. coli sacculus, with one notable exception: the C-1 carboxyl group of the D-isoglutamyl residue is partially amidated with N-acetylputrescine. Cyanelles harbor on their completely sequenced 135.6-kb genome genes for approximately 150 polypeptides, many of which are nucleus encoded in higher plants. Nevertheless, there are striking parallels in genome organization between cyanelles (and other primitive plastids) and higher plant chloroplasts. The transit sequences of nucleus-encoded cyanelle preproteins resemble stroma targeting peptides of higher plant chloroplast precursors. Heterologous import of precursors from C. paradoxa into isolated pea chloroplasts is possible and vice versa. Cyanelles are considered to represent a very early, diverging branch of plastid evolution and are derived from the semiautonomous endosymbiont that had already abandoned about 90% of its genetic information but still retained its prokaryotic wall. Recent data on the molecular biology of cyanelles and rhodoplasts are consistent with the assumption of a primary endosymbiotic event that was not only monophyletic with respect to the cyanobacterial invader, but also singular.

Cyanophora paradoxa is the best-investigated member of the glaucocystophyceae, phototrophic protists containing cyanelles, that is, plastids stabilized by a peptidoglycan-containing envelope. The classification of this group, comprising only eight (mostly monotypic) genera, is also based on parallels in morphology and organization of the “host cells” (Kies, 1992). Recently, this was corroborated by 16S and 18S rRNA-based phylogenetic analysis (Helmchen et al., 1995; Bhattacharya et al, 1995). Apart from C. paradoxa, only Glaucocystis nostochinearum can be grown at a reasonable rate. Thus, biochemical and molecular genetic data are mostly available for C. paradoxa and more precisely for the isolate 555UTEX (Pringsheim) that is kept in the major culture collections of algae. Biochemical work done on C. paradoxa and the sequencing of individual cyanelle genes have been described in several recent reviews (Schenk, 1992; Löffelhardt and Bohnert, 1994a,b). Here we discuss three topics: the cyanelle wall, aspects deduced from the complete cyanelle genome sequence, and protein translocation into and within cyanelles.     

Rubisco genes indicate a close phylogenetic relation between the plastids of Chromophyta and Rhodophyta

The genes for both subunits of Rubisco (rbcL, rbcS) are located on the plastome of the brown alga Ectocarpus siliculosus (Chromophyta, Phaeophyceae). The organization of these genes in the form of an operon was similar to that found in rhodoplasts, cyanobacteria and the plastids of Cryptomonas . Sequence analysis of the complete operon revealed a high degree of homology and great structural similarities to corresponding genes from two red algae. In contrast, sequence homology to Rubisco genes from chloroplasts and cyanobacteria was much lower. This clearly indicated a close phylogenetic relationship between the plastids of Rhodophyta and Chromophyta which seem to have evolved independently from the chloroplasts (polyphyletic origin). Our data suggest that the plastids of Chromophyta and Cryptophyta have originated from endosymbiotic unicellular red algae. Surprisingly, red and brown algal Rubiscos show a significantly higher degree of homology to that from a hydrogen bacterium than to those from cyanobacteria.     

The complete chloroplast genome of the chlorarachniophyte Bigelowiella natans: evidence for independent origins of chlorarachniophyte and euglenid secondary endosymbionts

Chlorarachniophytes are amoeboflagellate cercozoans that acquired a plastid by secondary endosymbiosis. Chlorarachniophytes are the last major group of algae for which there is no completely sequenced plastid genome. Here we describe the 69.2-kbp chloroplast genome of the model chlorarachniophyte Bigelowiella natans. The genome is highly reduced in size compared with plastids of other photosynthetic algae and is closer in size to genomes of several nonphotosynthetic plastids. Unlike nonphotosynthetic plastids, however, the B. natans chloroplast genome has not sustained a massive loss of genes, and it retains nearly all of the functional photosynthesis-related genes represented in the genomes of other green algae. Instead, the genome is highly compacted and gene dense. The genes are organized with a strong strand bias, and several unusual rearrangements and inversions also characterize the genome; notably, an inversion in the small-subunit rRNA gene, a translocation of 3 genes in the major ribosomal protein operon, and the fragmentation of the cluster encoding the large photosystem proteins PsaA and PsaB. The chloroplast endosymbiont is known to be a green alga, but its evolutionary origin and relationship to other primary and secondary green plastids has been much debated. A recent hypothesis proposes that the endosymbionts of chlorarachniophytes and euglenids share a common origin (the Cabozoa hypothesis). We inferred phylogenies using individual and concatenated gene sequences for all genes in the genome. Concatenated gene phylogenies show a relationship between the B. natans plastid and the ulvophyte-trebouxiophyte-chlorophyte clade of green algae to the exclusion of Euglena. The B. natans plastid is thus not closely related to that of Euglena, which suggests that plastids originated independently in these 2 groups and the Cabozoa hypothesis is false.     

Plastid genome sequence of the cryptophyte alga Rhodomonas salina CCMP1319: lateral transfer of putative DNA replication machinery and a test of chromist plastid phylogeny

Cryptophytes are a group of unicellular algae with chlorophyll c-containing plastids derived from the uptake of a secondary (i.e., eukaryotic) endosymbiont. Biochemical and molecular data indicate that cryptophyte plastids are derived from red algae, yet the question of whether or not cryptophytes acquired their red algal plastids independent of those in heterokont, haptophyte, and dinoflagellate algae is of long-standing debate. To better understand the origin and evolution of the cryptophyte plastid, we have sequenced the plastid genome of Rhodomonas salina CCMP1319: at 135,854 bp, it is the largest secondary plastid genome characterized thus far. It also possesses interesting features not seen in the distantly related cryptophyte Guillardia theta or in other red secondary plastids, including pseudogenes, introns, and a bacterial-derived gene for the tau/gamma subunit of DNA polymerase III (dnaX), the first time putative DNA replication machinery has been found encoded in any plastid genome. Phylogenetic analyses indicate that dnaX was acquired by lateral gene transfer (LGT) in an ancestor of Rhodomonas, most likely from a firmicute bacterium. A phylogenomic survey revealed no additional cases of LGT, beyond a noncyanobacterial type rpl36 gene similar to that recently characterized in other cryptophytes and haptophytes. Rigorous concatenated analysis of 45 proteins encoded in 15 complete plastid genomes produced trees in which the heterokont, haptophyte, and cryptophyte (i.e., chromist) plastids were monophyletic, and heterokonts and haptophytes were each other's closest relatives. However, statistical support for chromist monophyly disappears when amino acids are recoded according to their chemical properties in order to minimize the impact of composition bias, and a significant fraction of the concatenate appears consistent with a sister-group relationship between cryptophyte and haptophyte plastids.     

Glycoprotein import: A common feature of complex plastids?

Complex plastids evolved by secondary endosymbiosis and are, in contrast to primary plastids, surrounded by 3 or 4 envelope membranes. Recently, we provided evidence that in diatoms proteins exist that get N-glycosylated during transport across the outermost membrane of the complex plastid. This gives rise to unique questions on the transport mechanisms of these bulky proteins, which get transported across up to 3 further membranes into the plastid stroma. Here we discuss our results in an evolutionary context and speculate about the existence of plastidal glycoproteins in other organisms with complex plastids.     

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.     

The origin of plastids

It is generally accepted that plastids first arose by acquisition of photosynthetic prokaryotic endosymbionts by non-photosynthetic eukaryotic hosts. It is also accepted that photosynthetic eukaryotes were acquired on several occasions as endosymbionts by non-photosynthetic eukaryote hosts to form secondary plastids. In some lineages, secondary plastids were lost and new symbionts were acquired, to form tertiary plastids. Most recent work has been interpreted to indicate that primary plastids arose only once, referred to as a 'monophyletic' origin. We critically assess the evidence for this. We argue that the combination of Ockham's razor and poor taxon sampling will bias studies in favour of monophyly. We discuss possible concerns in phylogenetic reconstruction from sequence data. We argue that improved understanding of lineage-specific substitution processes is needed to assess the reliability of sequence-based trees. Improved understanding of the timing of the radiation of present-day cyanobacteria is also needed. We suggest that acquisition of plastids is better described as the result of a process rather than something occurring at a discrete time, and describe the 'shopping bag' model of plastid origin. We argue that dinoflagellates and other lineages provide evidence in support of this.     

Hatena arenicola gen. et sp. nov., a katablepharid undergoing probable plastid acquisition

Hatena arenicola gen. et sp. nov., an enigmatic flagellate of the katablepharids, is described. It shows ultrastructural affinities to the katablepharids, including large and small ejectisomes, cell covering, and a feeding apparatus. Although molecular phylogenies of the 18S ribosomal DNA support its classification into the katablepharids, the cell is characterized by a dorsiventrally compressed cell shape and a crawling motion, both of which are unusual within this group. The most distinctive feature of Hatena arenicola is that it harbors a Nephroselmis symbiont. This symbiosis is distinct from previously reported cases of ongoing symbiosis in that the symbiont plastid is selectively enlarged, while other structures such as the mitochondria, Golgi body, cytoskeleton, and endomembrane system are degraded; the host and symbiont have developed a morphological association, i.e., the eyespot of the symbiont is always at the cell apex of Hatena arenicola; and only one daughter cell inherits the symbiont during cell division, resulting in a symbiont-bearing green cell and a symbiont-lacking colorless cell. Interestingly, the colorless cells have a feeding apparatus that corresponds to the location of the eyespot in symbiont-bearing cells, and they are able to feed on prey cells. This indicates that the morphology of the host depends on the presence or absence of the symbiont. These observations suggest that Hatena arenicola has a unique "half-plant, half-predator" life cycle; one cell divides into an autotrophic cell possessing a symbiotic Nephroselmis species, and a symbiont-lacking colorless cell, which later develops a feeding apparatus de novo. The evolutionary implications of Hatena arenicola as an intermediate step in plastid acquisition are discussed in the context of other examples of ongoing endosymbioses in dinoflagellates.