Metabolic trade-offs constrain the cell size ratio in a nitrogen-fixing symbiosis.

Biological dinitrogen (N2) fixation is a key metabolic process exclusively performed by prokaryotes, some of which are symbiotic with eukaryotes. Species of the marine haptophyte algae Braarudosphaera bigelowii harbor the N2-fixing endosymbiotic cyanobacteria UCYN-A, which might be evolving organelle-like characteristics. We found that the size ratio between UCYN-A and their hosts is strikingly conserved across sublineages/species, which is consistent with the size relationships of organelles in this symbiosis and other species. Metabolic modeling showed that this size relationship maximizes the coordinated growth rate based on trade-offs between resource acquisition and exchange. Our findings show that the size relationships of N2-fixing endosymbionts and organelles in unicellular eukaryotes are constrained by predictable metabolic underpinnings and that UCYN-A is, in many regards, functioning like a hypothetical N2-fixing organelle (or nitroplast).

New plastids, old proteins: repeated endosymbiotic acquisitions in kareniacean dinoflagellates.

Dinoflagellates are a diverse group of ecologically significant micro-eukaryotes that can serve as a model system for plastid symbiogenesis due to their susceptibility to plastid loss and replacement via serial endosymbiosis. Kareniaceae harbor fucoxanthin-pigmented plastids instead of the ancestral peridinin-pigmented ones and support them with a diverse range of nucleus-encoded plastid-targeted proteins originating from the haptophyte endosymbiont, dinoflagellate host, and/or lateral gene transfers (LGT). Here, we present predicted plastid proteomes from seven distantly related kareniaceans in three genera (Karenia, Karlodinium, and Takayama) and analyze their evolutionary patterns using automated tree building and sorting. We project a relatively limited ( ~ 10%) haptophyte signal pointing towards a shared origin in the family Chrysochromulinaceae. Our data establish significant variations in the functional distributions of these signals, emphasizing the importance of micro-evolutionary processes in shaping the chimeric proteomes. Analysis of plastid genome sequences recontextualizes these results by a striking finding the extant kareniacean plastids are in fact not all of the same origin, as two of the studied species (Karlodinium armiger, Takayama helix) possess plastids from different haptophyte orders than the rest.

Heteroplasmy Is Rare in Plant Mitochondria Compared with Plastids despite Similar Mutation Rates.

Plant cells harbor two membrane-bound organelles containing their own genetic material-plastids and mitochondria. Although the two organelles coexist and coevolve within the same plant cells, they differ in genome copy number, intracellular organization, and mode of segregation. How these attributes affect the time to fixation or, conversely, loss of neutral alleles is currently unresolved. Here, we show that mitochondria and plastids share the same mutation rate, yet plastid alleles remain in a heteroplasmic state significantly longer compared with mitochondrial alleles. By analyzing genetic variants across populations of the marine flowering plant Zostera marina and simulating organelle allele dynamics, we examine the determinants of allele segregation and allele fixation. Our results suggest that the bottlenecks on the cell population, e.g. during branching or seeding, and stratification of the meristematic tissue are important determinants of mitochondrial allele dynamics. Furthermore, we suggest that the prolonged plastid allele dynamics are due to a yet unknown active plastid partition mechanism. The dissimilarity between plastid and mitochondrial novel allele fixation at different levels of organization may manifest in differences in adaptation processes. Our study uncovers fundamental principles of organelle population genetics that are essential for further investigations of long-term evolution and molecular dating of divergence events.

Ecological Predictors of Organelle Genome Evolution: Phylogenetic Correlations with Taxonomically Broad, Sparse, Unsystematized Data.

Comparative analysis of variables across phylogenetically linked observations can reveal mechanisms and insights in evolutionary biology. As the taxonomic breadth of the sample of interest increases, challenges of data sparsity, poor phylogenetic resolution, and complicated evolutionary dynamics emerge. Here, we investigate a cross-eukaryotic question where all these problems exist: which organismal ecology features are correlated with gene retention in mitochondrial and chloroplast DNA (organelle DNA or oDNA). Through a wide palette of synthetic control studies, we first characterize the specificity and sensitivity of a collection of parametric and non-parametric phylogenetic comparative approaches to identify relationships in the face of such sparse and awkward datasets. This analysis is not directly focused on oDNA, and so provides generalizable insights into comparative approaches with challenging data. We then combine and curate ecological data coupled to oDNA genome information across eukaryotes, including a new semi-automated approach for gathering data on organismal traits from less systematized open-access resources including encyclopedia articles on species and taxa. The curation process also involved resolving several issues with existing datasets, including enforcing the clade-specificity of several ecological features and fixing incorrect annotations. Combining this unique dataset with our benchmarked comparative approaches, we confirm support for several known links between organismal ecology and organelle gene retention, identify several previously unidentified relationships constituting possible ecological contributors to oDNA genome evolution, and provide support for a recently hypothesized link between environmental demand and oDNA retention. We, with caution, discuss the implications of these findings for organelle evolution and of this pipeline for broad comparative analyses in other fields.

Reconstruction of Plastid Proteomes of Apicomplexans and Close Relatives Reveals the Major Evolutionary Outcomes of Cryptic Plastids.

Apicomplexans and related lineages comprise many obligate symbionts of animals; some of which cause notorious diseases such as malaria. They evolved from photosynthetic ancestors and transitioned into a symbiotic lifestyle several times, giving rise to species with diverse non-photosynthetic plastids. Here, we sought to reconstruct the evolution of the cryptic plastids in the apicomplexans, chrompodellids, and squirmids (ACS clade) by generating five new single-cell transcriptomes from understudied gregarine lineages, constructing a robust phylogenomic tree incorporating all ACS clade sequencing datasets available, and using these to examine in detail, the evolutionary distribution of all 162 proteins recently shown to be in the apicoplast by spatial proteomics in Toxoplasma. This expanded homology-based reconstruction of plastid proteins found in the ACS clade confirms earlier work showing convergence in the overall metabolic pathways retained once photosynthesis is lost, but also reveals differences in the degrees of plastid reduction in specific lineages. We show that the loss of the plastid genome is common and unexpectedly find many lineage- and species-specific plastid proteins, suggesting the presence of evolutionary innovations and neofunctionalizations that may confer new functional and metabolic capabilities that are yet to be discovered in these enigmatic organelles.

Cellular and environmental dynamics influence species-specific extents of organelle gene retention.

Mitochondria and plastids rely on many nuclear-encoded genes, but retain small subsets of the genes they need to function in their own organelle DNA (oDNA). Different species retain different numbers of oDNA genes, and the reasons for these differences are not completely understood. Here, we use a mathematical model to explore the hypothesis that the energetic demands imposed by an organism's changing environment influence how many oDNA genes it retains. The model couples the physical biology of cell processes of gene expression and transport to a supply-and-demand model for the environmental dynamics to which an organism is exposed. The trade-off between fulfilling metabolic and bioenergetic environmental demands, and retaining genetic integrity, is quantified for a generic gene encoded either in oDNA or in nuclear DNA. Species in environments with high-amplitude, intermediate-frequency oscillations are predicted to retain the most organelle genes, whereas those in less dynamic or noisy environments the fewest. We discuss support for, and insight from, these predictions with oDNA data across eukaryotic taxa, including high oDNA gene counts in sessile organisms exposed to day-night and intertidal oscillations (including plants and algae) and low counts in parasites and fungi.

Complete genome of a nonphotosynthetic cyanobacterium in a diatom reveals recent adaptations to an intracellular lifestyle.

The evolution of mitochondria and plastids from bacterial endosymbionts were key events in the origin and diversification of eukaryotic cells. Although the ancient nature of these organelles makes it difficult to understand the earliest events that led to their establishment, the study of eukaryotic cells with recently evolved obligate endosymbiotic bacteria has the potential to provide important insight into the transformation of endosymbionts into organelles. Diatoms belonging to the family Rhopalodiaceae and their endosymbionts of cyanobacterial origin (i.e., "spheroid bodies") are emerging as a useful model system in this regard. The spheroid bodies, which appear to enable rhopalodiacean diatoms to use gaseous nitrogen, became established after the divergence of extant diatom families. Here we report what is, to our knowledge, the first complete genome sequence of a spheroid body, that of the rhopalodiacean diatom Epithemia turgida. The E. turgida spheroid body (EtSB) genome was found to possess a gene set for nitrogen fixation, as anticipated, but is reduced in size and gene repertoire compared with the genomes of their closest known free-living relatives. The presence of numerous pseudogenes in the EtSB genome suggests that genome reduction is ongoing. Most strikingly, our genomic data convincingly show that the EtSB has lost photosynthetic ability and is metabolically dependent on its host cell, unprecedented characteristics among cyanobacteria, and cyanobacterial symbionts. The diatom-spheroid body endosymbiosis is thus a unique system for investigating the processes underlying the integration of a bacterial endosymbiont into eukaryotic cells.

Differential remodelling of peroxisome function underpins the environmental and metabolic adaptability of diplonemids and kinetoplastids.

The remodelling of organelle function is increasingly appreciated as a central driver of eukaryotic biodiversity and evolution. Kinetoplastids including Trypanosoma and Leishmania have evolved specialized peroxisomes, called glycosomes. Glycosomes uniquely contain a glycolytic pathway as well as other enzymes, which underpin the physiological flexibility of these major human pathogens. The sister group of kinetoplastids are the diplonemids, which are among the most abundant eukaryotes in marine plankton. Here we demonstrate the compartmentalization of gluconeogenesis, or glycolysis in reverse, in the peroxisomes of the free-living marine diplonemid, Diplonema papillatum Our results suggest that peroxisome modification was already under way in the common ancestor of kinetoplastids and diplonemids, and raise the possibility that the central importance of gluconeogenesis to carbon metabolism in the heterotrophic free-living ancestor may have been an important selective driver. Our data indicate that peroxisome modification is not confined to the kinetoplastid lineage, but has also been a factor in the success of their free-living euglenozoan relatives.

The ancient and widespread nature of the ER-mitochondria encounter structure.

Mitochondria are the result of a billion years of integrative evolution, converting a once free-living bacterium to an organelle deeply linked to diverse cellular processes. One way in which mitochondria are integrated with nonendosymbiotically derived organelles is via endoplasmic reticulum (ER)-mitochondria contact sites. The ER membrane is physically tethered to the mitochondrial outer membrane by the ER-mitochondria encounter structure (ERMES). However, to date, ERMES has only ever been found in the fungal lineage. Here, we bioinformatically demonstrate that ERMES is present in lineages outside Fungi and validate this inference by mass spectrometric identification of ERMES components in Acanthamoeba castellanii mitochondria. We further demonstrate that ERMES is retained in hydrogenosome-bearing but not mitosome-bearing organisms, yielding insight into the process of reductive mitochondrial evolution. Finally, we find that the taxonomic distribution of ERMES is most consistent with rooting the eukaryotic tree between Amorphea (Animals + Fungi + Amoebozoa) + Excavata and all other eukaryotes (Diaphoratickes).

Reverse transcription of spliced psbA mRNA in Chlamydomonas spp. and its possible role in evolutionary intron loss.

Reverse transcription of mRNA is thought to be an important first step in a model that explains certain evolutionary changes within genes, such as the loss of introns or RNA editing sites. In this model, reverse transcription of mRNA produces cDNA molecules that replace part of the parental gene by homologous recombination. In vivo evidence of reverse transcription of physiologically relevant mRNAs is generally lacking, however, except in genetically engineered cells. Here, we provide in vivo evidence for reverse transcription of the chloroplast psbA mRNA in two naturally occurring species of Chlamydomonas (raudensis and subcaudata) that is based on the presence of spliced cDNAs in both organisms. The psbA cDNAs, which lack the group II intron of the genomic gene, are nearly full length, and the majority of them--though not all--are in the form of RNA-cDNA hybrids. Moreover, the presence in these species of psbA cDNAs is correlated with the loss of an early group I intron from the same psbA gene. The group II intron that interrupts psbA in C. raudensis and C. subcaudata potentially encodes a protein with a reverse transcriptase domain, and the C. raudensis protein was shown to have reverse transcriptase activity in vitro. These results provide strong evidence for reverse transcription of a physiologically important mRNA (psbA) in two species of Chlamydomonas that have also lost an intron from the same gene, possibly through recombination with the cDNA.

Organelle evolution, fragmented rRNAs, and Carl.

I am honored to have been asked to contribute to this memorial issue, although I cannot claim to have known Carl Woese well. Carl's insights and the discoveries that his research group made over the years certainly stimulated my own research program, and at several points early on, interactions with him were pivotal in my career. Here I comment on these personal dealings with Carl and emphasize his influence in two areas of long-standing interest in my lab: organelle evolution and rRNA evolution.

Remarkable kinetoplast, cytostome-cytopharynx complex, and storage-related structures as dissected by three-dimensional reconstruction of Trypanosoma sp. 858 isolated from a toad (Amphibia: Anura).

In Brazil, the Trypanosoma sp. 858 was isolated from a toad (Anura: Bufonidae: Rhinella ictericus) and successfully maintained in cultures. We previously demonstrated that this trypanosome is different but tightly clustered phylogenetically with other trypanosomes from anurans. In this study, we addressed the ultrastructural features of cultured epimastigotes of this new trypanosome. Our results showed very long and thin free motile forms exhibiting a long flagellum and remarkable large and loose K-DNA network. In addition, the anterior portion contained many acidocalcisomes and a well-developed spongiome tubules-contractile vacuole system. One of the main morphological features of this anuran trypanosome was the presence of a complex cytostome-cytopharynx with a specialized membrane coating at the entrance, which is often hidden by the flagellum. Other conspicuous features are the presence of lipid-like droplets, lamellar membrane limited inclusions, and one very large reservosome, all at the posterior portion of the cell body. This new trypanosome may constitute an excellent model for organelles studies related to endocytosis and lipid storage, as demonstrated herein using scanning and transmission electron microscopy and three-dimensional models obtained by either electron microscopy tomography or dual-beam slice and view series.

Phylogenomics Identifies a New Major Subgroup of Apicomplexans, Marosporida class nov., with Extreme Apicoplast Genome Reduction.

The phylum Apicomplexa consists largely of obligate animal parasites that include the causative agents of human diseases such as malaria. Apicomplexans have also emerged as models to study the evolution of nonphotosynthetic plastids, as they contain a relict chloroplast known as the apicoplast. The apicoplast offers important clues into how apicomplexan parasites evolved from free-living ancestors and can provide insights into reductive organelle evolution. Here, we sequenced the transcriptomes and apicoplast genomes of three deep-branching apicomplexans, Margolisiella islandica, Aggregata octopiana, and Merocystis kathae. Phylogenomic analyses show that these taxa, together with Rhytidocystis, form a new lineage of apicomplexans that is sister to the Coccidia and Hematozoa (the lineages including most medically significant taxa). Members of this clade retain plastid genomes and the canonical apicomplexan plastid metabolism. However, the apicoplast genomes of Margolisiella and Rhytidocystis are the most reduced of any apicoplast, are extremely GC-poor, and have even lost genes for the canonical plastidial RNA polymerase. This new lineage of apicomplexans, for which we propose the class Marosporida class nov., occupies a key intermediate position in the apicomplexan phylogeny, and adds a new complexity to the models of stepwise reductive evolution of genome structure and organelle function in these parasites.

Genomic analysis of the tryptome reveals molecular mechanisms of gland cell evolution.

BACKGROUND: Understanding the drivers of morphological diversity is a persistent challenge in evolutionary biology. Here, we investigate functional diversification of secretory cells in the sea anemone Nematostella vectensis to understand the mechanisms promoting cellular specialization across animals. RESULTS: We demonstrate regionalized expression of gland cell subtypes in the internal ectoderm of N. vectensis and show that adult gland cell identity is acquired very early in development. A phylogenetic survey of trypsins across animals suggests that this gene family has undergone numerous expansions. We reveal unexpected diversity in trypsin protein structure and show that trypsin diversity arose through independent acquisitions of non-trypsin domains. Finally, we show that trypsin diversification in N. vectensis was effected through a combination of tandem duplication, exon shuffling, and retrotransposition. CONCLUSIONS: Together, these results reveal the numerous evolutionary mechanisms that drove trypsin duplication and divergence during the morphological specialization of cell types and suggest that the secretory cell phenotype is highly adaptable as a vehicle for novel secretory products.

The Symbiosome: Legume and Rhizobia Co-evolution toward a Nitrogen-Fixing Organelle?

In legume nodules, symbiosomes containing endosymbiotic rhizobial bacteria act as temporary plant organelles that are responsible for nitrogen fixation, these bacteria develop mutual metabolic dependence with the host legume. In most legumes, the rhizobia infect post-mitotic cells that have lost their ability to divide, although in some nodules cells do maintain their mitotic capacity after infection. Here, we review what is currently known about legume symbiosomes from an evolutionary and developmental perspective, and in the context of the different interactions between diazotroph bacteria and eukaryotes. As a result, it can be concluded that the symbiosome possesses organelle-like characteristics due to its metabolic behavior, the composite origin and differentiation of its membrane, the retargeting of host cell proteins, the control of microsymbiont proliferation and differentiation by the host legume, and the cytoskeletal dynamics and symbiosome segregation during the division of rhizobia-infected cells. Different degrees of symbiosome evolution can be defined, specifically in relation to rhizobial infection and to the different types of nodule. Thus, our current understanding of the symbiosome suggests that it might be considered a nitrogen-fixing link in organelle evolution and that the distinct types of legume symbiosomes could represent different evolutionary stages toward the generation of a nitrogen-fixing organelle.