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Nonphotosynthetic plastids retain important biological functions and are indispensable for cell viability. However, the detailed processes underlying the loss of plastidal functions other than photosynthesis remain to be fully understood. In this study, we used transcriptomics, subcellular localization, and phylogenetic analyses to characterize the biochemical complexity of the nonphotosynthetic plastids of the apochlorotic diatom Nitzschia sp. NIES-3581. We found that these plastids have lost isopentenyl pyrophosphate biosynthesis and ribulose-1,5-bisphosphate carboxylase/oxygenase-based carbon fixation but have retained various proteins for other metabolic pathways, including amino acid biosynthesis, and a portion of the Calvin-Benson cycle comprised only of glycolysis/gluconeogenesis and the reductive pentose phosphate pathway (rPPP). While most genes for plastid proteins involved in these reactions appear to be phylogenetically related to plastid-targeted proteins found in photosynthetic relatives, we also identified a gene that most likely originated from a cytosolic protein gene. Based on organellar metabolic reconstructions of Nitzschia sp. NIES-3581 and the presence/absence of plastid sugar phosphate transporters, we propose that plastid proteins for glycolysis, gluconeogenesis, and rPPP are retained even after the loss of photosynthesis because they feed indispensable substrates to the amino acid biosynthesis pathways of the plastid. Given the correlated retention of the enzymes for plastid glycolysis, gluconeogenesis, and rPPP and those for plastid amino acid biosynthesis pathways in distantly related nonphotosynthetic plastids and cyanobacteria, we suggest that this substrate-level link with plastid amino acid biosynthesis is a key constraint against loss of the plastid glycolysis/gluconeogenesis and rPPP proteins in multiple independent lineages of nonphotosynthetic algae/plants.

The origin of land plants from algae is a long-standing question in evolutionary biology. It is becoming increasingly clear that many characters that were once assumed to be 'embryophyte specific' can in fact be found in their closest algal relatives, the streptophyte algae. One such case is the phenylpropanoid pathway. While biochemical data indicate that streptophyte algae harbor lignin-like components, the phenylpropanoid core pathway, which serves as the backbone of lignin biosynthesis, has been proposed to have arisen at the base of the land plants. Here we revisit this hypothesis using a wealth of new sequence data from streptophyte algae. Tracing the biochemical pathway towards lignin biogenesis, we show that most of the genes required for phenylpropanoid synthesis and the precursors for lignin production were already present in streptophyte algae. Nevertheless, phylogenetic analyses and protein structure predictions of one of the key enzyme classes in lignin production, cinnamyl alcohol dehydrogenase (CAD), suggest that CADs of streptophyte algae are more similar to sinapyl alcohol dehydrogenases (SADs). This suggests that the end-products of the pathway leading to lignin biosynthesis in streptophyte algae may facilitate the production of lignin-like compounds and defense molecules. We hypothesize that streptophyte algae already possessed the genetic toolkit from which the capacity to produce lignin later evolved in vascular plants.

Blastocystis spp. are the most prevalent eukaryotic microbes found in the intestinal tract of humans. Here we present an in-depth investigation of lateral gene transfer (LGT) in the genome of Blastocystis sp. subtype 1. Using rigorous phylogeny-based methods and strict validation criteria, we show that ∼2.5% of the genes of this organism were recently acquired by LGT. We identify LGTs both from prokaryote and eukaryote donors. Several transfers occurred specifically in ancestors of a subset of Blastocystis subtypes, demonstrating that LGT is an ongoing process. Functional predictions reveal that these genes are involved in diverse metabolic pathways, many of which appear related to adaptation of Blastocystis to the gut environment. Specifically, we identify genes involved in carbohydrate scavenging and metabolism, anaerobic amino acid and nitrogen metabolism, oxygen-stress resistance, and pH homeostasis. A number of the transferred genes encoded secreted proteins that are potentially involved in infection, escaping host defense, or most likely affect the prokaryotic microbiome and the inflammation state of the gut. We also show that Blastocystis subtypes differ in the nature and copy number of LGTs that could relate to variation in their prevalence and virulence. Finally, we identified bacterial-derived genes encoding NH3-dependent nicotinamide adenine dinucleotide (NAD) synthase in Blastocystis and other protozoan parasites, which are promising targets for drug development. Collectively, our results suggest new avenues for research into the role of Blastocystis in intestinal disease and unequivocally demonstrate that LGT is an important mechanism by which eukaryotic microbes adapt to new environments.

Members of the genus Paramoeba (including Neoparamoeba) (Amoebozoa) are single-celled eukaryotes of economic and ecological importance because of their association with disease in a variety of marine animals including fish, sea urchins, and lobster. Interestingly, they harbor a eukaryotic endosymbiont of kinetoplastid ancestry, Perkinsela sp. To investigate the complex relationship between Paramoeba spp. and Perkinsela sp., as well as the relationships between different Paramoeba species, molecular data was obtained for four novel isolates. We also acquired new data from the urchin pathogen P. invadens. Comprehensive molecular phylogenetic analyses were carried out using 33 newly obtained 18S rDNA sequences from the host amoebae and 16 new 18S rDNA sequences from their corresponding Perkinsela sp., together with all publicly available 18S molecular data. Intra-isolate 18S rDNA nucleotide diversity was found to be surprisingly high within the various species of Paramoeba, but relatively low within their Perkinsela sp. endosymbionts. 18S rDNA phylogenies and ParaFit co-evolution analysis revealed a high degree of congruence between the Paramoeba and Perkinsela sp. tree topologies, strongly suggesting that a single endosymbiotic event occurred in the common ancestor of known Paramoeba species, and that the endosymbionts have been inherited vertically ever since.

Plastids evolved from cyanobacteria by endosymbiosis. During the course of evolution, the coding capacity of plastid genomes shrinks due to gene loss or transfer to the nucleus. In the green lineage, however, there were apparent gene gains including that of ycf1. Although its function is still debated, YCF1 has proven to be a useful marker for plastid evolution. YCF1 sequence and predicted structural features unite the plastid genomes of land plants with those of their closest algal relatives, the higher streptophyte algae; YCF1 appears to have undergone pronounced changes during the course of streptophyte algal evolution. Using new data, we show that YCF1 underwent divergent evolution in the common ancestor of higher streptophyte algae and Klebsormidiophycae. This divergence resulted in the origin of an extreme, klebsormidiophycean-specific YCF1 and the higher streptophyte Ste-YCF1. Most importantly, our analysis uncovers a conserved carboxy-terminal sequence stretch within YCF1 that is unique to higher streptophytes and hints at an important, yet unexplored function.

Cyanobacteria occupy a special place in the pantheon of prokaryotic life. It is in the ancestors of these ubiquitous microbes that oxygenic photosynthesis first evolved more than 2 billion y ago (1), and it is from endosymbiotic cyanobacteria that the plastids (chloroplasts) of plants and algae are derived (2). Modern-day cyanobacteria are diverse in form and function; they include coccoid marine picoplankton such as Prochlorococcus (3), freshwater biofilm-forming genera [e.g., Gloeomargarita (4)], and filamentous taxa capable of fixing nitrogen [e.g., Nostoc (5)]. In PNAS, Nakayama et al. (6) add an exciting chapter to the story of cyanobacterial diversity. The authors describe the genome sequence of a cyanobacterium living ectosymbiotically on an eye-catching dinoflagellate named Ornithocercus magnificus . Their results provide insight into the nature of an enigmatic symbiotic relationship and reveal the existence of a cryptic, globally distributed cyanobacterial lineage that has until now gone unappreciated. Ornithocercus is indeed magnificent, even by dinoflagellate standards. The surface of this heterotrophic marine protist is decorated with crown-shaped, cellulosic outcroppings that extend from the cell body in different directions (Fig. 1) (7). The “upper” crown forms an extracellular chamber in which autofluorescent cyanobacteria reside, and microscopic evidence suggests that the bacteria can be vertically transmitted from mother to daughter chambers during host cell division (8). First observed more than 100 y ago (9), the biology of these so-called phaeosomes has long been mysterious. What type of cyanobacterium are they? How far do they roam? What is the nature of their interactions with the dinoflagellate host? Fig. 1. Light micrograph of an Ornithocercus dinoflagellate. The ectosymbiotic OmCyn cyanobacteria are visible within an extracellular chamber on the “upper” crown of the cell. Image courtesy of Takuro Nakayama (Tohoku University, Sendai, … [↵][1]1Email: jmarchib{at}dal.ca. [1]: #xref-corresp-1-1

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. Electronic supplementary material The online version of this article (10.1186/s12915-018-0593-5) contains supplementary material, which is available to authorized users.

Protists, which are single-celled eukaryotes, critically influence the ecology and chemistry of marine ecosystems, but genome-based studies of these organisms have lagged behind those of other microorganisms. However, recent transcriptomic studies of cultured species, complemented by meta-omics analyses of natural communities, have increased the amount of genetic information available for poorly represented branches on the tree of eukaryotic life. This information is providing insights into the adaptations and interactions between protists and other microorganisms and macroorganisms, but many of the genes sequenced show no similarity to sequences currently available in public databases. A better understanding of these newly discovered genes will lead to a deeper appreciation of the functional diversity and metabolic processes in the ocean. In this Review, we summarize recent developments in our understanding of the ecology, physiology and evolution of protists, derived from transcriptomic studies of cultured strains and natural communities, and discuss how these novel large-scale genetic datasets will be used in the future.

Summary The kinetoplastids are a widespread and important group of single-celled eukaryotes, many of which are devastating parasites of animals, including humans [1–3]. We have discovered a new insect trypanosomatid in the gut of Culex pipiens mosquitoes. Glyceraldehyde-3-phosphate dehydrogenase- and SSU rRNA-based phylogenetic analyses show this parasite to constitute a distinct branch between the free-living Bodo saltans and the obligatory parasitic clades represented by the genus Trypanosoma and other trypanosomatids. From draft genome sequence data, we identified 114 protein genes shared among the new flagellate, 15 trypanosomatid species, B. saltans , and the heterolobosean Naegleria gruberi , as well as 129 protein genes shared with the basal kinetoplastid Perkinsela sp. Individual protein phylogenies together with analyses of concatenated alignments show that the new species, here named Paratrypanosoma confusum n. gen., n. sp., branches with very high support at the base of the family Trypanosomatidae. P. confusum thus represents a long-sought-after missing link between the ancestral free-living bodonids and the derived parasitic trypanosomatids. Further analysis of the P. confusum genome should provide insight into the emergence of parasitism in the medically important trypanosomatids.

Summary An amoeba named Paulinella harbours ‘chromatophores', cyanobacterium-derived photosynthetic bodies that evolved independent of plastids. Proteomics has shown that hundreds of nucleus-encoded proteins are targeted to the chromatophore, revealing the host cell's contributions to its recently established organelle.

de Vries and Archibald introduce the topic of plastid genomes - prokaryotic genomes housed within eukaryotic algae and plants.

The concept of symbiosis – defined in 1879 by de Bary as ‘the living together of unlike organisms’ – has a rich and convoluted history in biology. In part, because it questioned the concept of the individual, symbiosis fell largely outside mainstream science and has traditionally received less attention than other research disciplines. This is gradually changing. In nature organisms do not live in isolation but rather interact with, and are impacted by, diverse beings throughout their life histories. Symbiosis is now recognized as a central driver of evolution across the entire tree of life, including, for example, bacterial endosymbionts that provide insects with vital nutrients and the mitochondria that power our own cells. Symbioses between microbes and their multicellular hosts also underpin the ecological success of some of the most productive ecosystems on the planet, including hydrothermal vents and coral reefs. In November 2017, scientists working in fields spanning the life sciences came together at a Company of Biologists’ workshop to discuss the origin, maintenance, and long-term implications of symbiosis from the complementary perspectives of cell biology, ecology, evolution and genomics, taking into account both model and non-model organisms. Here, we provide a brief synthesis of the fruitful discussions that transpired., Summary: At a recent Company of Biologists workshop, evolutionary biologists discussed the major outstanding questions in symbiosis research.

Understanding plant evolution and diversity in a phylogenomic context is an enormous challenge due, in part, to limited availability of genome-scale data across phylodiverse species. The 10KP (10,000 Plants) Genome Sequencing Project will sequence and characterize representative genomes from every major clade of embryophytes, green algae, and protists (excluding fungi) within the next 5 years. By implementing and continuously improving leading-edge sequencing technologies and bioinformatics tools, 10KP will catalogue the genome content of plant and protist diversity and make these data freely available as an enduring foundation for future scientific discoveries and applications. 10KP is structured as an international consortium, open to the global community, including botanical gardens, plant research institutes, universities, and private industry. Our immediate goal is to establish a policy framework for this endeavor, the principles of which are outlined here.

Background The Synurophyceae is one of most important photosynthetic stramenopile algal lineages in freshwater ecosystems. They are characterized by siliceous scales covering the cell or colony surface and possess plastids of red-algal secondary or tertiary endosymbiotic origin. Despite their ecological and evolutionary significance, the relationships amongst extant Synurophyceae are unclear, as is their relationship to most other stramenopiles. Results Here we report a comparative analysis of plastid genomes sequenced from five representative synurophycean algae. Most of these plastid genomes are highly conserved with respect to genome structure and coding capacity, with the exception of gene re-arrangements and partial duplications at the boundary of the inverted repeat and single-copy regions. Several lineage-specific gene loss/gain events and intron insertions were detected (e.g., cemA, dnaB, syfB, and trnL). Conclusions Unexpectedly, the cemA gene of Synurophyceae shows a strong relationship with sequences from members of the green-algal lineage, suggesting the occurrence of a lateral gene transfer event. Using a molecular clock approach based on silica fossil record data, we infer the timing of genome re-arrangement and gene gain/loss events in the plastid genomes of Synurophyceae. Electronic supplementary material The online version of this article (10.1186/s12862-018-1316-9) contains supplementary material, which is available to authorized users.

Plastids stem from free-living cyanobacteria. The transition from endosymbiont to organelle involved strong reductive evolution. Modern-day plastid genomes possess only a small fraction of the genes present in their cyanobacterial progenitors. In addition to genome reduction, plastids underwent modifications that facilitated recruitment of host-derived proteins and metabolites; both processes contributed to organellogenesis and a shift in control over plastid function from the organellar genome to that of the host. It is likely that most of the modifications to the early plastid happened before the major radiations that led to today's algae and plants. Plastids nevertheless exhibit substantial variation in form and function. In this chapter, we highlight some of the evolutionary implications of the differences in the genetic capacities of plastids across the breadth of plant and algal diversity. We focus on the transition from genetic semiautonomy, which is of relevance in the context of the endosymbiotic spread of plastids and kleptoplasty, to the high degree of nuclear control over plastid function seen in land plants. Genomic and transcriptomic investigations of diverse plants and algae have revealed important differences in the coding capacity of plastid genomes in different lineages, raising questions about how the plastid's own genetic capabilities impact its physiology as well as that of its host.

The internal compartmentation of eukaryotic cells not only allows separation of biochemical processes but it also creates the requirement for systems that can selectively transport proteins across the membrane boundaries. Although most proteins function in a single subcellular compartment, many are able to enter two or more compartments, a phenomenon known as dual or multiple targeting. The aminoacyl-tRNA synthetases (aaRSs), which catalyze the ligation of tRNAs to their cognate amino acids, are particularly prone to functioning in multiple subcellular compartments. They are essential for translation, so they are required in every compartment where translation takes place. In diatoms, there are three such compartments, the plastid, the mitochondrion, and the cytosol. In cryptophytes, translation also takes place in the periplastid compartment (PPC), which is the reduced cytoplasm of the plastid’s red algal ancestor and which retains a reduced red algal nucleus. We searched the organelle and nuclear genomes of the cryptophyte Guillardia theta and the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana for aaRS genes and found an insufficient number of genes to provide each compartment with a complete set of aaRSs. We therefore inferred, with support from localization predictions, that many aaRSs are dual targeted. We tested four of the predicted dual targeted aaRSs with green fluorescent protein fusion localizations in P. tricornutum and found evidence for dual targeting to the mitochondrion and plastid in P. tricornutum and G. theta, and indications for dual targeting to the PPC and cytosol in G. theta. This is the first report of dual targeting in diatoms or cryptophytes.

Background Cryptophytes are an ecologically important group of algae comprised of phototrophic, heterotrophic and osmotrophic species. This lineage is of great interest to evolutionary biologists because their plastids are of red algal secondary endosymbiotic origin. Cryptophytes have a clear phylogenetic affinity to heterotrophic eukaryotes and possess four genomes: host-derived nuclear and mitochondrial genomes, and plastid and nucleomorph genomes of endosymbiotic origin. Results To gain insight into cryptophyte mitochondrial genome evolution, we sequenced the mitochondrial DNAs of five species and performed a comparative analysis of seven genomes from the following cryptophyte genera: Chroomonas, Cryptomonas, Hemiselmis, Proteomonas, Rhodomonas, Storeatula and Teleaulax. The mitochondrial genomes were similar in terms of their general architecture, gene content and presence of a large repeat region. However, gene order was poorly conserved. Characteristic features of cryptophyte mtDNAs included large syntenic clusters resembling α-proteobacterial operons that encode bacteria-like rRNAs, tRNAs, and ribosomal protein genes. The cryptophyte mitochondrial genomes retain almost all genes found in many other eukaryotes including the nad, sdh, cox, cob, and atp genes, with the exception of sdh2 and atp3. In addition, gene cluster analysis showed that cryptophytes possess a gene order closely resembling the jakobid flagellates Jakoba and Reclinomonas. Interestingly, the cox1 gene of R. salina, T. amphioxeia, and Storeatula species was found to contain group II introns encoding a reverse transcriptase protein, as did the cob gene of Storeatula species CCMP1868. Conclusions These newly sequenced genomes increase the breadth of data available from algae and will aid in the identification of general trends in mitochondrial genome evolution. While most of the genomes were highly conserved, extensive gene arrangements have shuffled gene order, perhaps due to genome rearrangements associated with hairpin-containing mobile genetic elements, tRNAs with palindromic sequences, and tandem repeat sequences. The cox1 and cob gene sequences suggest that introns have recently been acquired during cryptophyte evolution. Comparison of phylogenetic trees based on plastid and mitochondrial genome data sets underscore the different evolutionary histories of the host and endosymbiont components of present-day cryptophytes. Electronic supplementary material The online version of this article (10.1186/s12864-018-4626-9) contains supplementary material, which is available to authorized users.

Contents Summary 1428 I. The singularity of plant terrestrialization 1428 II. Adaptation vs exaptation - what shaped the land plant toolkit? 1430 III. Trait mosaicism in (higher-branching) streptophyte algae 1431 IV. CONCLUSIONS a streptophyte algal perspective on land plant trait evolution 1432 Acknowledgements 1432 ORCID 1433 References 1433 SUMMARY: Photosynthetic eukaryotes thrive anywhere there is sunlight and water. But while such organisms are exceptionally diverse in form and function, only one phototrophic lineage succeeded in rising above its substrate: the land plants (embryophytes). Molecular phylogenetic data show that land plants evolved from streptophyte algae most closely related to extant Zygnematophyceae, and one of the principal aims of plant evolutionary biology is to uncover the key features of such algae that enabled this important transition. At the present time, however, mosaic and reductive evolution blur our picture of the closest algal ancestors of plants. Here we discuss recent progress and problems in inferring the biology of the algal progenitor of the terrestrial photosynthetic macrobiome.

Endosymbiotic relationships between eukaryotic and prokaryotic cells are common in nature. Endosymbioses between two eukaryotes are also known; cyanobacterium-derived plastids have spread horizontally when one eukaryote assimilated another. A unique instance of a non-photosynthetic, eukaryotic endosymbiont involves members of the genus Paramoeba, amoebozoans that infect marine animals such as farmed fish and sea urchins. Paramoeba species harbor endosymbionts belonging to the Kinetoplastea, a diverse group of flagellate protists including some that cause devastating diseases. To elucidate the nature of this eukaryote-eukaryote association, we sequenced the genomes and transcriptomes of Paramoeba pemaquidensis and its endosymbiont Perkinsela sp. The endosymbiont nuclear genome is ~9.5 Mbp in size, the smallest of a kinetoplastid thus far discovered. Genomic analyses show that Perkinsela sp. has lost the ability to make a flagellum but retains hallmark features of kinetoplastid biology, including polycistronic transcription, trans-splicing, and a glycosome-like organelle. Mosaic biochemical pathways suggest extensive ‘cross-talk’ between the two organisms, and electron microscopy shows that the endosymbiont ingests amoeba cytoplasm, a novel form of endosymbiont-host communication. Our data reveal the cell biological and biochemical basis of the obligate relationship between Perkinsela sp. and its amoeba host, and provide a foundation for understanding pathogenicity determinants in economically important Paramoeba.

DNA sequencing is faster and cheaper than ever before but quantity does not necessarily mean quality. Towards a comprehensive understanding of the microbial biosphere, we need more reference genomes from single-celled eukaryotes (protists) across the full breadth of eukaryotic diversity.

Photosynthesis evolved in eukaryotes by the endosymbiosis of a cyanobacterium, the future plastid, within a heterotrophic host. This primary endosymbiosis occurred in the ancestor of Archaeplastida, a eukaryotic supergroup that includes glaucophytes, red algae, green algae and land plants [1–4]. However, while the endosymbiotic origin of plastids from a single cyanobacterial ancestor is firmly established, the nature of that ancestor remains controversial: plastids have been proposed to derive from either early- or late-branching cyanobacterial lineages [5–11]. To solve this issue, we carried out phylogenomic and super-network analyses of the most comprehensive dataset analyzed so far including plastid-encoded proteins and nucleus-encoded proteins of plastid origin resulting from endosymbiotic gene transfer (EGT) of primary photosynthetic eukaryotes, as well as wide-ranging genome data from cyanobacteria, including novel lineages. Our analyses strongly support that plastids evolved from deep-branching cyanobacteria and that the present-day closest cultured relative of primary plastids is Gloeomargarita lithophora. This species belongs to a recently discovered cyanobacterial lineage widespread in freshwater microbialites and microbial mats [12, 13]. The ecological distribution of this lineage sheds new light on the environmental conditions where the emergence of photosynthetic eukaryotes occurred, most likely in a terrestrial-freshwater setting. The fact that glaucophytes, the first archaeplastid lineage to diverge, are exclusively found in freshwater ecosystems reinforces this hypothesis. Therefore, plastids not only emerged early within cyanobacteria, but the first photosynthetic eukaryotes likely evolved in terrestrial-freshwater settings, not in oceans as commonly thought.

Eukaryotic cells evolved the ability to harness the energy of sunlight by the process of endosymbiosis. Well after the endosymbiotic origin of mitochondria, an ancestor of modern-day cyanobacteria was taken up by a heterotrophic eukaryote and gradually became a fully integrated subcellular organelle within the cytoplasm of its host. This event led to the differentiation of three algal lineages: red algae, glaucophyte algae, and green algae (as well as their land plant derivatives), all of which possess double membrane-bound plastids. On multiple occasions after these three lineages diverged, their plastids were acquired by other eukaryotes by the process of secondary endosymbiosis, that is, the engulfment and retention of a primary plastid-bearing alga by a second, nonphotosynthetic eukaryote host. Secondary plastids are characterized by the presence of either three or four outer membranes and, in some cases, the retention of the nucleus of the algal endosymbiont in a residual cytoplasmic compartment. This article gives a detailed overview of the genetic, molecular, and cell biological aspects of secondary endosymbiosis, summarizes the diversity of secondary endosymbioses, and reviews what is known about the tempo and mode of secondary endosymbiosis and its role in the diversification of photosynthetic life. The phenomenon of tertiary endosymbiosis is also discussed.

Environmental molecular sequence surveys have opened a window on the hidden riches of the microbial biosphere. Recent genetic 'barcoding' and single-cell genomics studies have provided a snapshot of the biology of diplonemids - abundant, diverse, marine heterotrophic protists whose ecological roles are becoming clearer.

John Archibald praises a compelling guide to the past 3 billion years — and its molecular historians. John Archibald praises a compelling guide to the past 3 billion years — and its molecular historians.

Reductive genome evolution is seen in organisms living in clo se association with each other, such as in endosymbiosis, symbiosis, and parasitism. The reduced genomes of endosymbionts and parasites often exhibit similar features such as high gene densities and A+ T compositional bias. Little is known about how the regulation of gene expression has been affected in organisms with highly compacted genomes. We studied gene transcription patterns in “nucleomorph” genomes, which are relic nuclear genomes of algal endosymbionts found in cryptophytes and chlorarachniophytes. We examined nuclear and nucleomorph gene transcription patterns using RNA-Seq transcriptome and genome mapping analyses in representatives of both lineages. In all four examined genomes, the most highly transcribed nucleomorph gene category was found to be plastid-associated genes. Remarkably, only 0.49–3.37% of the nucleomorph genomes of these organisms did not have any mRNA counterpart in our RNA-Seq data sets, and nucleomorph genes show equal or higher levels of transcription than their counterparts in the nuclear genomes. We hypothesize that elevated levels of nucleomorph gene transcription may serve to counteract the degradation or modification of protein function due to the loss of interacting proteins in the nucleomorph and nucleomorph-associated subcellular compartments.

Organelle DNA is no stranger to palindromic repeats. But never has a mitochondrial or plastid genome been described in which every coding region is part of a distinct palindromic unit. While sequencing the mitochondrial DNA of the nonphotosynthetic green alga Polytomella magna, we uncovered precisely this type of genic arrangement. The P. magna mitochondrial genome is linear and made up entirely of palindromes, each containing 1–7 unique coding regions. Consequently, every gene in the genome is duplicated and in an inverted orientation relative to its partner. And when these palindromic genes are folded into putative stem-loops, their predicted translational start sites are often positioned in the apex of the loop. Gel electrophoresis results support the linear, 28-kb monomeric conformation of the P. magna mitochondrial genome. Analyses of other Polytomella taxa suggest that palindromic mitochondrial genes were present in the ancestor of the Polytomella lineage and lost or retained to various degrees in extant species. The possible origins and consequences of this bizarre genomic architecture are discussed.

Chlorarachniophytes are unicellular marine algae with plastids (chloroplasts) of secondary endosymbiotic origin. Chlorarachniophyte cells retain the remnant nucleus (nucleomorph) and cytoplasm (periplastidial compartment, PPC) of the green algal endosymbiont from which their plastid was derived. To characterize the diversity of nucleus-encoded proteins targeted to the chlorarachniophyte plastid, nucleomorph, and PPC, we isolated plastid–nucleomorph complexes from the model chlorarachniophyte Bigelowiella natans and subjected them to high-pressure liquid chromatography-tandem mass spectrometry. Our proteomic analysis, the first of its kind for a nucleomorph-bearing alga, resulted in the identification of 324 proteins with 95% confidence. Approximately 50% of these proteins have predicted bipartite leader sequences at their amino termini. Nucleus-encoded proteins make up >90% of the proteins identified. With respect to biological function, plastid-localized light-harvesting proteins were well represented, as were proteins involved in chlorophyll biosynthesis. Phylogenetic analyses revealed that many, but by no means all, of the proteins identified in our proteomic screen are of apparent green algal ancestry, consistent with the inferred evolutionary origin of the plastid and nucleomorph in chlorarachniophytes.

Background Kinetoplastea is a diverse protist lineage composed of several of the most successful parasites on Earth, organisms whose metabolisms have coevolved with those of the organisms they infect. Parasitic kinetoplastids have emerged from free-living, non-pathogenic ancestors on multiple occasions during the evolutionary history of the group. Interestingly, in both parasitic and free-living kinetoplastids, the heme pathway—a core metabolic pathway in a wide range of organisms—is incomplete or entirely absent. Indeed, Kinetoplastea investigated thus far seem to bypass the need for heme biosynthesis by acquiring heme or intermediate metabolites directly from their environment. Results Here we report the existence of a near-complete heme biosynthetic pathway in Perkinsela spp., kinetoplastids that live as obligate endosymbionts inside amoebozoans belonging to the genus Paramoeba/Neoparamoeba. We also use phylogenetic analysis to infer the evolution of the heme pathway in Kinetoplastea. Conclusion We show that Perkinsela spp. is a deep-branching kinetoplastid lineage, and that lateral gene transfer has played a role in the evolution of heme biosynthesis in Perkinsela spp. and other Kinetoplastea. We also discuss the significance of the presence of seven of eight heme pathway genes in the Perkinsela genome as it relates to its endosymbiotic relationship with Paramoeba. Electronic supplementary material The online version of this article (doi:10.1186/s12862-016-0664-6) contains supplementary material, which is available to authorized users.

Cryptophytes are unicellular, biflagellate algae with plastids (chloroplasts) derived from the uptake of a red algal endosymbiont. These organisms are unusual in that the nucleus of the engulfed red alga persists in a highly reduced form called a nucleomorph. Nucleomorph genomes are remarkable in their small size (1,000 kilobase pairs [kbp]) and high degree of compaction (∼1 kbp per gene). Here, we investigated the molecular and karyotypic diversity of nucleomorph genomes in members of the genus Cryptomonas. 18S rDNA genes were amplified, sequenced, and analyzed from C. tetrapyrenoidosa Skuja CCAP979/63, C. erosa Ehrenb. emmend. Hoef-Emden CCAP979/67, Cryptomonas sp. CCAP979/52, C. lundii Hoef-Emden et Melkonian CCAP979/69, and C. lucens Skuja CCAP979/35 in the context of a large set of publicly available nucleomorph 18S rDNA sequences. Pulsed-field gel electrophoresis (PFGE) was used to examine the nucleomorph genome karyotype of each of these strains. Individual chromosomes ranged from ∼160 to 280 kbp in size, with total genome sizes estimated to be ∼600-655 kbp. Unexpectedly, the nucleomorph karyotype of Cryptomonas sp. CCAP979/52 is significantly different from that of C. tetrapyrenoidosa and C. lucens, despite the fact that their 18S rDNA genes are99% identical to one another. These results suggest that nucleomorph karyotype similarity is not a reliable indicator of evolutionary affinity and provides a starting point for further investigation of the fine-scale dynamics of nucleomorph genome evolution within members of the genus Cryptomonas.

Cryptomonads are a ubiquitous and diverse assemblage of aquatic flagellates. The relatively obscure genus Hemiselmis includes some of the smallest of these cells. This genus contained only two species until 1967, when Butcher described seven new marine species mainly on the basis of observations with the light microscope. However, from these seven taxa, only H. amylifera and H. oculata were validly published. Additionally, the features Butcher used to distinguish species have since been questioned, and the taxonomy within Hemiselmis has remained clouded due to the difficulty in unambiguously applying his classification and validating many of his species. As a result, marine strains are often placed into one of three species-H. rufescens Parke, H. virescens Droop, or the invalid H. brunnescens Butcher-based on cell color alone. Here we applied microscopic and molecular tools to 13 publicly available Hemiselmis strains in an effort to clarify species boundaries. SEM failed to provide sufficient morphological variation to distinguish species of Hemiselmis, and results from LM did not correlate with clades found using both molecular phylogenetic and nucleomorph genome karyotype analysis, indicating a high degree of morphological plasticity within species. On the basis of molecular characters and collection geography we recognize four new marine species of Hemiselmis-H. cryptochromatica sp. nov., H. andersenii sp. nov., H. pacifica sp. nov., and H. tepida sp. nov.-from the waters around North America.

Chlorarachniophyte algae possess four DNA-containing compartments per cell, the nucleus, mitochondrion, plastid and nucleomorph, the latter being a relic nucleus derived from a secondary endosymbiont. While the evolutionary dynamics of plastid and nucleomorph genomes have been investigated, a comparative investigation of mitochondrial genomes (mtDNAs) has not been carried out. We have sequenced the complete mtDNA of Lotharella oceanica and compared it to that of another chlorarachniophyte, Bigelowiella natans. The linear mtDNA of L. oceanica is 36.7 kbp in size and contains 35 protein genes, three rRNAs and 24 tRNAs. The codons GUG and UUG appear to be capable of acting as initiation codons in the chlorarachniophyte mtDNAs, in addition to AUG. Rpl16, rps4 and atp8 genes are missing in L.oceanica mtDNA, despite being present in B. natans mtDNA. We searched for and found, mitochondrial rpl16 and rps4 genes with spliceosomal introns in the L. oceanica nuclear genome, indicating that mitochondrion-to-host-nucleus gene transfer occurred after the divergence of these two genera. Despite being of similar size and coding capacity, the level of synteny between L. oceanica and B. natans mtDNA is low, suggesting frequent rearrangements. Overall, our results suggest that chlorarachniophyte mtDNAs are more evolutionarily dynamic than their plastid counterparts.

Cryptophytes are a diverse lineage of marine and freshwater, photosynthetic and secondarily nonphotosynthetic algae that acquired their plastids (chloroplasts) by “secondary” (i.e., eukaryote–eukaryote) endosymbiosis. Consequently, they are among the most genetically complex cells known and have four genomes: a mitochondrial, plastid, “master” nuclear, and residual nuclear genome of secondary endosymbiotic origin, the so-called “nucleomorph” genome. Sequenced nucleomorph genomes are ∼1,000-kilobase pairs (Kbp) or less in size and are comprised of three linear, compositionally biased chromosomes. Although most functionally annotated nucleomorph genes encode proteins involved in core eukaryotic processes, up to 40% of the genes in these genomes remain unidentifiable. To gain insight into the function and evolutionary fate of nucleomorph genomes, we used 454 and Illumina technologies to completely sequence the nucleomorph genome of the cryptophyte Chroomonas mesostigmatica CCMP1168. At 702.9 Kbp in size, the C. mesostigmatica nucleomorph genome is the largest and the most complex nucleomorph genome sequenced to date. Our comparative analyses reveal the existence of a highly conserved core set of genes required for maintenance of the cryptophyte nucleomorph and plastid, as well as examples of lineage-specific gene loss resulting in differential loss of typical eukaryotic functions, e.g., proteasome-mediated protein degradation, in the four cryptophyte lineages examined.

Planctomycetes, Verrucomicrobia and Chlamydia are prokaryotic phyla, sometimes grouped together as the PVC superphylum of eubacteria. Some PVC species possess interesting attributes, in particular, internal membranes that superficially resemble eukaryotic endomembranes. Some biologists now claim that PVC bacteria are nucleus-bearing prokaryotes and are considered evolutionary intermediates in the transition from prokaryote to eukaryote. PVC prokaryotes do not possess a nucleus and are not intermediates in the prokaryote-to-eukaryote transition. Here we summarise the evidence that shows why all of the PVC traits that are currently cited as evidence for aspiring eukaryoticity are either analogous (the result of convergent evolution), not homologous, to eukaryotic traits; or else they are the result of horizontal gene transfers.

Summary Complex cellular machines and processes are commonly believed to be products of selection, and it is typically understood to be the job of evolutionary biologists to show how selective advantage can account for each step in their origin and subsequent growth in complexity. Here, we describe how complex machines might instead evolve in the absence of positive selection through a process of ‘‘presuppression,’’ first termed constructive neutral evolution (CNE) more than a decade ago. If an autonomously functioning cellular component acquires mutations that make it dependent for function on another, preexisting component or process, and if there are multiple ways in which such dependence may arise, then dependence inevitably will arise and reversal to independence is unlikely. Thus, CNE is a unidirectional evolutionary ratchet leading to complexity, if complexity is equated with the number of components or steps necessary to carry out a cellular process. CNE can explain ‘‘functions’’ that seem to make little sense in terms of cellular economy, like RNA editing or splicing, but it may also contribute to the complexity of machines with clear benefit to the cell, like the ribosome, and to organismal complexity overall. We suggest that CNE-based evolutionary scenarios are in these and other cases less forced than the selectionist or adaptationist narratives that are generally told. 2011 IUBMB IUBMB Life, 63(7): 528–537, 2011

The year 1970 saw the publication of Origin of Eukaryotic Cells by Lynn Margulis. This influential book brought the exciting and weighty problems of cellular evolution to the scientific mainstream, simultaneously breaking new ground and ‘re-discovering’ the decades-old ideas of German and Russian biologists. In this commemorative review, I discuss the 40 years that have elapsed since this landmark publication, with a focus on the ‘molecular era’: how DNA sequencing and comparative genomics have proven beyond all doubt the central tenets of the endosymbiont hypothesis for the origin of mitochondria and plastids, and, at the same time, revealed a genetic and genomic complexity in modern-day eukaryotes that could not have been imagined in decades past.

The use of molecular methods is altering our understanding of the microbial biosphere and the complexity of the tree of life. Here, we report a newly discovered uncultured plastid-bearing eukaryotic lineage named the rappemonads. Phylogenies using near-complete plastid ribosomal DNA (rDNA) operons demonstrate that this group represents an evolutionarily distinct lineage branching with haptophyte and cryptophyte algae. Environmental DNA sequencing revealed extensive diversity at North Atlantic, North Pacific, and European freshwater sites, suggesting a broad ecophysiology and wide habitat distribution. Quantitative PCR analyses demonstrate that the rappemonads are often rare but can form transient blooms in the Sargasso Sea, where high 16S rRNA gene copies mL −1 were detected in late winter. This pattern is consistent with these microbes being a member of the rare biosphere, whose constituents have been proposed to play important roles under ecosystem change. Fluorescence in situ hybridization revealed that cells from this unique lineage were 6.6 ± 1.2 × 5.7 ± 1.0 μm, larger than numerically dominant open-ocean phytoplankton, and appear to contain two to four plastids. The rappemonads are unique, widespread, putatively photosynthetic algae that are absent from present-day ecosystem models and current versions of the tree of life.

Nucleomorphs are the remnant nuclei of algal endosymbionts that were engulfed by nonphotosynthetic host eukaryotes. These peculiar organelles are found in cryptomonad and chlorarachniophyte algae, where they evolved from red and green algal endosymbionts, respectively. Despite their independent origins, cryptomonad and chlorarachniophyte nucleomorph genomes are similar in size and structure: they are both

Chlorarachniophytes are enigmatic marine unicellular algae that acquired photosynthesis by secondary endosymbiosis. Chlorarachniophytes are unusual in that the nucleus of the engulfed algal cell (a green alga) persists in a miniaturized form, termed a nucleomorph. The nucleomorph genome of the model chlorarachniophyte, Bigelowiella natans CCMP621, is 373 kilobase pairs (kbp) in size, the smallest nuclear genome characterized to date. The B. natans nucleomorph genome is composed of three chromosomes, each with canonical eukaryotic telomeres and sub-telomeric ribosomal DNA (rDNA) operons transcribed away from the chromosome end. Here we present the complete rDNA operon and telomeric region from the nucleomorph genome of Lotharella oceanica CCMP622, a newly characterized chlorarachniophyte strain with a genome ∼610 kbp in size, significantly larger than all other known chlorarachniophytes. We show that the L. oceanica rDNA operon is in the opposite chromosomal orientation to that of B. natans. Furthermore, we determined the rDNA operon orientation of five additional chlorarachniophyte strains, the majority of which possess the same arrangement as L. oceanica, with the exception of Chlorarachnion reptans and those very closely related to B. natans. It is thus possible that the ancestral rDNA operon orientation of the chlorarachniophyte nucleomorph genome might have been the same as in the independently evolved, red algal-derived, nucleomorph genomes of cryptophytes. A U2 small nuclear RNA gene was found adjacent to the telomere in Gymnochlora stellata CCMP2057 and Chlorarachnion sp. CCMP2014. This feature may represent a useful evolutionary character for inferring the relationships among extant lineages.

Here we present evidence for a complex evolutionary history of actin genes in red algae and cryptomonads, a group that acquired photosynthesis secondarily through the engulfment of a red algal endosymbiont. Four actin genes were found in the nuclear genome of the cryptomonad, Guillardia theta, and in the genome of the red alga, Galdieria sulphuraria, a member of the Cyanidiophytina. Phylogenetic analyses reveal that the both organisms possess two distinct sequence types, designated “type-1” and “type-2.” A weak but consistent phylogenetic affinity between the cryptomonad type-2 sequences and the type-2 sequences of G. sulphuraria and red algae belonging to the Rhodophytina was observed. This is consistent with the possibility that the cryptomonad type-2 sequences are derived from the red algal endosymbiont that gave rise to the cryptomonad nucleomorph and plastid. Red algae as a whole possess two very different actin sequence types, with G. sulphuraria being the only organism thus far known to possess both. The common ancestor of Rhodophytina and Cyanidiophytina may have had two actin genes, with differential loss explaining the distribution of these genes in modern-day groups. Our study provides new insight into the evolution and divergence of actin genes in cryptomonads and red algae, and in doing so underscores the challenges associated with heterogeneity in actin sequence evolution and ortholog/paralog detection.

Petalomonas sphagnophila is a poorly studied plastid-lacking euglenid flagellate living in Sphagnum-dominated peatlands. Here we present a broad-ranging microscopic, molecular and microspectrophotometric analysis of uncultured P. sphagnophila collected from four field locations in Nova Scotia, Canada. Consistent with its morphological characteristics, 18S ribosomal DNA (rDNA) phylogenies indicate that P. sphagnophila is specifically related to Petalomonas cantuscygni, the only other Petalomonas species sequenced to date. One of the peculiar characteristics of P. sphagnophila is the presence of several green-pigmented particles approximately 5 mum in diameter in its cytoplasm, which a previously published study suggested to be cyanobacterial endosymbionts. New data presented here, however, suggest that the green intracellular body may not be a cyanobacterium but rather an uncharacterized prokaryote yet to be identified by molecular sequencing. 16S rDNA library sequencing and fluorescence in situ hybridizations show that P. sphagnophila also harbors several other endobionts, including bacteria that represent five novel genus-level groups (one firmicute and four different proteobacteria). 16S rDNA phylogenies suggest that three of these endobionts are related to obligate intracellular bacteria such as Rickettsiales and Coxiella, while the others are related to the Daphnia pathogen Spirobacillus cienkowskii or belong to the Thermoactinomycetaceae. TEM, 16S rDNA library sequencing and a battery of PCR experiments show that the presence of the five P. sphagnophila endobionts varies markedly among the four geographic collections and even among individuals collected from the same location but at different time points. Our study adds significantly to the growing evidence for complex and dynamic protist-bacterial associations in nature.

Nucleomorphs are the remnant nuclei of algal endosymbionts in cryptophytes and chlorarachniophytes, two evolutionarily distinct unicellular eukaryotic lineages that acquired photosynthesis secondarily by the engulfment of red and green algae, respectively. At less than one million base pairs in size, nucleomorph genomes are the most highly reduced nuclear genomes known, with three small linear chromosomes and a gene density similar to that seen in prokaryotes. The independent origin of nucleomorphs in cryptophytes and chlorarachniophytes presents an interesting opportunity to study the reductive evolutionary forces that have led to their remarkable convergence upon similar genome architectures and coding capacities. In this article, we review the current state of knowledge with respect to the structure, function, origin, and evolution of nucleomorph genomes across the known diversity of cryptophyte and chlorarachniophyte algae.

A patchily distributed gene family is often taken as evidence for horizontal gene transfer (HGT) events, but it may also result solely from multiple gene losses. The RJL family of uncharacterised Ras-like GTPases was previously suggested to have undergone HGT events between protists and deuterostome metazoans, owing to the apparent absence of RJL in intermediate groups (Nepomuceno-Silva, J.L., de Melo, L.D., Mendonca, S.M., Paixao, J.C., Lopes, U.G., 2004. RJLs: a new family of Ras-related GTP-binding proteins. Gene 327, 221–232). We have reanalysed the phylogenetic distribution and phylogeny of the RJL family, taking advantage of the recent expansion of sequence data available from diverse eukaryotes. We found that RJL orthologs are much more widely distributed than previously assumed. At least one representative encoding an RJL protein could be identified for each of the six major eukaryotic “supergroups” (Opisthokonta, Amoebozoa, Excavata, Archaeplastida, Chromalveolata, and Rhizaria) as well as for a species of Apusomonadida, a deep lineage that may not be specifically related to any of the recognized supergroups. Phylogenetic analyses do not support HGT of RJL genes between the major eukaryotic lineages, indicating that the RJL family was present in the last eukaryotic common ancestor and was lost several times over the course of eukaryotic evolution. Interestingly, RJL was lost from all taxa lacking flagellated cells and from a few lineages that build structurally unusual or reduced flagella, raising the intriguing possibility that RJL proteins are functionally associated with the flagellar apparatus. The RJL GTPase domain has been fused with the DnaJ domain on two separate occasions: in the Holozoa (before the split of Metazoa and choanoflagellates), giving rise to the previously known Rbj type of RJL with the DnaJ domain at the C-terminus, and independently in Alveolata resulting in novel proteins with the DnaJ domain at the N-terminus. These independent fusions suggest that RJL proteins may generally function via regulating the DnaJ–Hsp70 module.

The cryptomonads are a group of unicellular algae that acquired photosynthesis through the engulfment of a red algal cell, a process called secondary endosymbiosis. Here, we present the complete plastid genome sequence of the secondarily nonphotosynthetic species Cryptomonas paramecium CCAP977/2a. The approximately 78 kilobase pair (Kbp) C. paramecium genome contains 82 predicted protein genes, 29 transfer RNA genes, and a single pseudogene (atpF). The C. paramecium plastid genome is approximately 50 Kbp smaller than those of the photosynthetic cryptomonads Guillardia theta and Rhodomonas salina; 71 genes present in the G. theta and/or R. salina plastid genomes are missing in C. paramecium. The pet, psa, and psb photosynthetic gene families are almost entirely absent. Interestingly, the ribosomal RNA operon, present as inverted repeats in most plastid genomes (including G. theta and R. salina), exists as a single copy in C. paramecium. The G + C content (38%) is higher in C. paramecium than in other cryptomonad plastid genomes, and C. paramecium plastid genes are characterized by significantly different codon usage patterns and increased evolutionary rates. The content and structure of the C. paramecium plastid genome provides insight into the changes associated with recent loss of photosynthesis in a predominantly photosynthetic group of algae and reveals features shared with the plastid genomes of other secondarily nonphotosynthetic eukaryotes.

Understanding the early evolution and diversification of eukaryotes relies on a fully resolved phylogenetic tree. In recent years, most eukaryotic diversity has been assigned to six putative supergroups, but the evolutionary origin of a few major “orphan” lineages remains elusive. Two ecologically important orphan groups are the heterotrophic Telonemia and Centroheliozoa. Telonemids have been proposed to be related to the photosynthetic cryptomonads or stramenopiles and centrohelids to haptophytes, but molecular phylogenies have failed to provide strong support for any phylogenetic hypothesis. Here, we investigate the origins of Telonema subtilis (a telonemid) and Raphidiophrys contractilis (a centrohelid) by large-scale 454 pyrosequencing of cDNA libraries and including new genomic data from two cryptomonads (Guillardia theta and Plagioselmis nannoplanctica) and a haptophyte (Imantonia rotunda). We demonstrate that 454 sequencing of cDNA libraries is a powerful and fast method of sampling a high proportion of protist genes, which can yield ample information for phylogenomic studies. Our phylogenetic analyses of 127 genes from 72 species indicate that telonemids and centrohelids are members of an emerging major group of eukaryotes also comprising cryptomonads and haptophytes. Furthermore, this group is possibly closely related to the SAR clade comprising stramenopiles (heterokonts), alveolates, and Rhizaria. Our results link two additional heterotrophic lineages to the predominantly photosynthetic chromalveolate supergroup, providing a new framework for interpreting the evolution of eukaryotic cell structures and the diversification of plastids.

Plants and algae acquired photosynthesis through the assimilation of a prokaryotic endosymbiont related to the ancestors of modern-day cyanobacteria. This landmark event, known as the primary endosymbiotic origin of plastids, is generally thought to have occurred only once during the history of eukaryotes and to have given rise to the plastids of green algae, land plants, red algae and glaucophyte algae through vertical evolution. Plastids have also spread horizontally across the tree of eukaryotes by “secondary” endosymbioses involving heterotrophic host eukaryotes and both green and red algal endosymbionts. Here I provide an overview of current research in the area of plastid evolution, focusing on the latest advances in the field of algal comparative genomics. Recent genome-scale analyses of both photosynthetic and non-photosynthetic eukaryotes have provided fresh new insight into the pattern and process of secondary endosymbiosis, although it is still not possible to discern with confidence the number of endosymbiotic events that gave rise to the known spectrum of eukaryotic phototrophs. In fact, with more genomic data has come the intriguing possibility that the nuclear genomes of some secondary plastid-containing algae are a mosaic of genes derived from multiple endosymbioses, adding yet another layer of complexity to the convoluted evolutionary history of these fascinating organisms.