The energetic costs of cellular complexity in evolution.

The evolutionary history of cells has been marked by drastic increases in complexity. Some hypothesize that such cellular complexification requires a massive energy flux as the origin of new features is hypothetically more energetically costly than their evolutionary maintenance. However, it remains unclear how increases in cellular complexity demand more energy. I propose that the early evolution of new genes with weak functions imposes higher energetic costs by overexpression before their functions are evolutionarily refined. In the long term, the accumulation of new genes deviates resources away from growth and reproduction. Accrued cellular complexity further requires additional infrastructure for its maintenance. Altogether, this suggests that larger and more complex cells are defined by increased survival but lower reproductive capacity.

A DRP-like pseudoenzyme coordinates with MICOS to promote cristae architecture.

Mitochondrial cristae architecture is crucial for optimal respiratory function of the organelle. Cristae shape is maintained in part by the mitochondrial inner membrane-localized MICOS complex. While MICOS is required for normal cristae morphology, the precise mechanistic role of each of the seven human MICOS subunits, and how the complex coordinates with other cristae shaping factors, has not been fully determined. Here, we examine the MICOS complex in Schizosaccharomyces pombe, a minimal model whose genome only encodes for four core subunits. Using an unbiased proteomics approach, we identify a poorly characterized inner mitochondrial membrane protein that interacts with MICOS and is required to maintain cristae morphology, which we name Mmc1. We demonstrate that Mmc1 works in concert with MICOS complexes to promote normal mitochondrial morphology and respiratory function. Bioinformatic analyses reveal that Mmc1 is a distant relative of the Dynamin-Related Protein (DRP) family of GTPases, which are well established to shape and remodel membranes. We find that, like DRPs, Mmc1 self-associates and forms high molecular weight assemblies. Interestingly, however, Mmc1 is a pseudoenzyme that lacks key residues required for GTP binding and hydrolysis, suggesting it does not dynamically remodel membranes. These data are consistent with a model in which Mmc1 stabilizes cristae architecture by acting as a scaffold to support cristae ultrastructure on the matrix side of the inner membrane. Our study reveals a new class of proteins that evolved early in fungal phylogeny and is required for the maintenance of cristae architecture. This highlights the possibility that functionally analogous proteins work with MICOS to establish cristae morphology in metazoans.

Single-Cell Genomics Reveals the Divergent Mitochondrial Genomes of Retaria (Foraminifera and Radiolaria).

Mitochondria originated from an ancient bacterial endosymbiont that underwent reductive evolution by gene loss and endosymbiont gene transfer to the nuclear genome. The diversity of mitochondrial genomes published to date has revealed that gene loss and transfer processes are ongoing in many lineages. Most well-studied eukaryotic lineages are represented in mitochondrial genome databases, except for the superphylum Retaria-the lineage comprising Foraminifera and Radiolaria. Using single-cell approaches, we determined two complete mitochondrial genomes of Foraminifera and two nearly complete mitochondrial genomes of radiolarians. We report the complete coding content of an additional 14 foram species. We show that foraminiferan and radiolarian mitochondrial genomes contain a nearly fully overlapping but reduced mitochondrial gene complement compared to other sequenced rhizarians. In contrast to animals and fungi, many protists encode a diverse set of proteins on their mitochondrial genomes, including several ribosomal genes; however, some aerobic eukaryotic lineages (euglenids, myzozoans, and chlamydomonas-like algae) have reduced mitochondrial gene content and lack all ribosomal genes. Similar to these reduced outliers, we show that retarian mitochondrial genomes lack ribosomal protein and tRNA genes, contain truncated and divergent small and large rRNA genes, and contain only 14 or 15 protein-coding genes, including nad1, -3, -4, -4L, -5, and -7, cob, cox1, -2, and -3, and atp1, -6, and -9, with forams and radiolarians additionally carrying nad2 and nad6, respectively. In radiolarian mitogenomes, a noncanonical genetic code was identified in which all three stop codons encode amino acids. Collectively, these results add to our understanding of mitochondrial genome evolution and fill in one of the last major gaps in mitochondrial sequence databases. IMPORTANCE We present the reduced mitochondrial genomes of Retaria, the rhizarian lineage comprising the phyla Foraminifera and Radiolaria. By applying single-cell genomic approaches, we found that foraminiferan and radiolarian mitochondrial genomes contain an overlapping but reduced mitochondrial gene complement compared to other sequenced rhizarians. An alternative genetic code was identified in radiolarian mitogenomes in which all three stop codons encode amino acids. Collectively, these results shed light on the divergent nature of the mitochondrial genomes from an ecologically important group, warranting further questions into the biological underpinnings of gene content variability and genetic code variation between mitochondrial genomes.

Intracytoplasmic-membrane development in alphaproteobacteria involves the homolog of the mitochondrial crista-developing protein Mic60.

Mitochondrial cristae expand the surface area of respiratory membranes and ultimately allow for the evolutionary scaling of respiration with cell volume across eukaryotes. The discovery of Mic60 homologs among alphaproteobacteria, the closest extant relatives of mitochondria, suggested that cristae might have evolved from bacterial intracytoplasmic membranes (ICMs). Here, we investigated the predicted structure and function of alphaproteobacterial Mic60, and a protein encoded by an adjacent gene Orf52, in two distantly related purple alphaproteobacteria, Rhodobacter sphaeroides and Rhodopseudomonas palustris. In addition, we assessed the potential physical interactors of Mic60 and Orf52 in R. sphaeroides. We show that the three α helices of mitochondrial Mic60's mitofilin domain, as well as its adjacent membrane-binding amphipathic helix, are present in alphaproteobacterial Mic60. The disruption of Mic60 and Orf52 caused photoheterotrophic growth defects, which are most severe under low light conditions, and both their disruption and overexpression led to enlarged ICMs in both studied alphaproteobacteria. We also found that alphaproteobacterial Mic60 physically interacts with BamA, the homolog of Sam50, one of the main physical interactors of eukaryotic Mic60. This interaction, responsible for making contact sites at mitochondrial envelopes, has been conserved in modern alphaproteobacteria despite more than a billion years of evolutionary divergence. Our results suggest a role for Mic60 in photosynthetic ICM development and contact site formation at alphaproteobacterial envelopes. Overall, we provide support for the hypothesis that mitochondrial cristae evolved from alphaproteobacterial ICMs and have therefore improved our understanding of the nature of the mitochondrial ancestor.

Purple photosymbioses.

Muñoz-Gómez and Hess introduce purple photosymbioses, which involve a heterotrophic protist host and anoxygenic photosymbionts from the phylum Proteobacteria.

Energetics and evolution of anaerobic microbial eukaryotes.

Mitochondria and aerobic respiration have been suggested to be required for the evolution of eukaryotic cell complexity. Aerobic respiration is several times more energetically efficient than fermentation. Moreover, aerobic respiration occurs at internalized mitochondrial membranes that are not constrained by a sublinear scaling with cell volume. However, diverse and complex anaerobic eukaryotes (for example, free-living and parasitic unicellular, and even small multicellular, eukaryotes) that exclusively rely on fermentation for energy generation have evolved repeatedly from aerobic ancestors. How do fermenting eukaryotes maintain their cell volumes and complexity while relying on such a low energy-yielding process? Here I propose that reduced rates of ATP generation in fermenting versus respiring eukaryotes are compensated for by longer cell cycles that satisfy lifetime energy demands. A literature survey and growth efficiency calculations show that fermenting eukaryotes divide approximately four to six times slower than aerobically respiring counterparts with similar cell volumes. Although ecological advantages such as competition avoidance offset lower growth rates and yields in the short term, fermenting eukaryotes inevitably have fewer physiological and ecological possibilities, which ultimately constrain their long-term evolutionary trajectories.

The role of mitochondrial energetics in the origin and diversification of eukaryotes.

The origin of eukaryotic cell size and complexity is often thought to have required an energy excess supplied by mitochondria. Recent observations show energy demands to scale continuously with cell volume, suggesting that eukaryotes do not have higher energetic capacity. However, respiratory membrane area scales superlinearly with the cell surface area. Furthermore, the consequences of the contrasting genomic architectures between prokaryotes and eukaryotes have not been precisely quantified. Here, we investigated (1) the factors that affect the volumes at which prokaryotes become surface area-constrained, (2) the amount of energy divested to DNA due to contrasting genomic architectures and (3) the costs and benefits of respiring symbionts. Our analyses suggest that prokaryotes are not surface area-constrained at volumes of 100‒103 µm3, the genomic architecture of extant eukaryotes is only slightly advantageous at genomes sizes of 106‒107 base pairs and a larger host cell may have derived a greater advantage (lower cost) from harbouring ATP-producing symbionts. This suggests that eukaryotes first evolved without the need for mitochondria since these ranges hypothetically encompass the last eukaryotic common ancestor and its relatives. Our analyses also show that larger and faster-dividing prokaryotes would have a shortage of respiratory membrane area and divest more energy into DNA. Thus, we argue that although mitochondria may not have been required by the first eukaryotes, eukaryote diversification was ultimately dependent on mitochondria.

Site-and-branch-heterogeneous analyses of an expanded dataset favour mitochondria as sister to known Alphaproteobacteria.

Determining the phylogenetic origin of mitochondria is key to understanding the ancestral mitochondrial symbiosis and its role in eukaryogenesis. However, the precise evolutionary relationship between mitochondria and their closest bacterial relatives remains hotly debated. The reasons include pervasive phylogenetic artefacts as well as limited protein and taxon sampling. Here we developed a new model of protein evolution that accommodates both across-site and across-branch compositional heterogeneity. We applied this site-and-branch-heterogeneous model (MAM60 + GFmix) to a considerably expanded dataset that comprises 108 mitochondrial proteins of alphaproteobacterial origin, and novel metagenome-assembled genomes from microbial mats, microbialites and sediments. The MAM60 + GFmix model fits the data much better and agrees with analyses of compositionally homogenized datasets with conventional site-heterogenous models. The consilience of evidence thus suggests that mitochondria are sister to the Alphaproteobacteria to the exclusion of MarineProteo1 and Magnetococcia. We also show that the ancestral presence of the crista-developing mitochondrial contact site and cristae organizing system (a mitofilin-domain-containing Mic60 protein) in mitochondria and the Alphaproteobacteria only supports their close relationship.

A microbial eukaryote with a unique combination of purple bacteria and green algae as endosymbionts.

Oxygenic photosynthesizers (cyanobacteria and eukaryotic algae) have repeatedly become endosymbionts throughout evolution. In contrast, anoxygenic photosynthesizers (e.g., purple bacteria) are exceedingly rare as intracellular symbionts. Here, we report on the morphology, ultrastructure, lifestyle, and metagenome of the only "purple-green" eukaryote known. The ciliate Pseudoblepharisma tenue harbors green algae and hundreds of genetically reduced purple bacteria. The latter represent a new candidate species of the Chromatiaceae that lost known genes for sulfur dissimilation. The tripartite consortium is physiologically complex because of the versatile energy metabolism of each partner but appears to be ecologically specialized as it prefers hypoxic sediments. The emergent niche of this complex symbiosis is predicted to be a partial overlap of each partners' niches and may be largely defined by anoxygenic photosynthesis and possibly phagotrophy. This purple-green ciliate thus represents an extraordinary example of how symbiosis merges disparate physiologies and allows emergent consortia to create novel ecological niches.

Constructive Neutral Evolution 20 Years Later.

Evolution has led to a great diversity that ranges from elegant simplicity to ornate complexity. Many complex features are often assumed to be more functional or adaptive than their simpler alternatives. However, in 1999, Arlin Stolzfus published a paper in the Journal of Molecular Evolution that outlined a framework in which complexity can arise through a series of non-adaptive steps. He called this framework Constructive Neutral Evolution (CNE). Despite its two-decade-old roots, many evolutionary biologists still appear to be unaware of this explanatory framework for the origins of complexity. In this perspective piece, we explain the theory of CNE and how it changes the order of events in narratives that describe the evolution of complexity. We also provide an extensive list of cellular features that may have become more complex through CNE. We end by discussing strategies to determine whether complexity arose through neutral or adaptive processes.

Independent accretion of TIM22 complex subunits in the animal and fungal lineages.

Background: The mitochondrial protein import complexes arose early in eukaryogenesis. Most of the components of the protein import pathways predate the last eukaryotic common ancestor. For example, the carrier-insertase TIM22 complex comprises the widely conserved Tim22 channel core. However, the auxiliary components of fungal and animal TIM22 complexes are exceptions to this ancient conservation. Methods: Using comparative genomics and phylogenetic approaches, we identified precisely when each TIM22 accretion occurred. Results: In animals, we demonstrate that Tim29 and Tim10b arose early in the holozoan lineage. Tim29 predates the metazoan lineage being present in the animal sister lineages, choanoflagellate and filastereans, whereas the erroneously named Tim10b arose from a duplication of Tim9 at the base of metazoans. In fungi, we show that Tim54 has representatives present in every holomycotan lineage including microsporidians and fonticulids, whereas Tim18 and Tim12 appeared much later in fungal evolution. Specifically, Tim18 and Tim12 arose from duplications of Sdh3 and Tim10, respectively, early in the Saccharomycotina. Surprisingly, we show that Tim54 is distantly related to AGK suggesting that AGK and Tim54 are extremely divergent orthologues and the origin of AGK/Tim54 interaction with Tim22 predates the divergence of animals and fungi. Conclusions: We argue that the evolutionary history of the TIM22 complex is best understood as the neutral structural divergence of an otherwise strongly functionally conserved protein complex. This view suggests that many of the differences in structure/subunit composition of multi-protein complexes are non-adaptive. Instead, most of the phylogenetic variation of functionally conserved molecular machines, which have been under stable selective pressures for vast phylogenetic spans, such as the TIM22 complex, is most likely the outcome of the interplay of random genetic drift and mutation pressure.

Genome-enabled phylogenetic and functional reconstruction of an araphid pennate diatom Plagiostriata sp. CCMP470, previously assigned as a radial centric diatom, and its bacterial commensal.

Diatoms are an ecologically fundamental and highly diverse group of algae, dominating marine primary production in both open-water and coastal communities. The diatoms include both centric species, which may have radial or polar symmetry, and the pennates, which include raphid and araphid species and arose within the centric lineage. Here, we use combined microscopic and molecular information to reclassify a diatom strain CCMP470, previously annotated as a radial centric species related to Leptocylindrus danicus, as an araphid pennate species in the staurosiroid lineage, within the genus Plagiostriata. CCMP470 shares key ultrastructural features with Plagiostriata taxa, such as the presence of a sternum with parallel striae, and the presence of a highly reduced labiate process on its valve; and this evolutionary position is robustly supported by multigene phylogenetic analysis. We additionally present a draft genome of CCMP470, which is the first genome available for a staurosiroid lineage. 270 Pfams (19%) found in the CCMP470 genome are not known in other diatom genomes, which otherwise does not hold big novelties compared to genomes of non-staurosiroid diatoms. Notably, our DNA library contains the genome of a bacterium within the Rhodobacterales, an alpha-proteobacterial lineage known frequently to associate with algae. We demonstrate the presence of commensal alpha-proteobacterial sequences in other published algal genome and transcriptome datasets, which may indicate widespread and persistent co-occurrence.

Homologue replacement in the import motor of the mitochondrial inner membrane of trypanosomes.

Many mitochondrial proteins contain N-terminal presequences that direct them to the organelle. The main driving force for their translocation across the inner membrane is provided by the presequence translocase-associated motor (PAM) which contains the J-protein Pam18. Here, we show that in the PAM of Trypanosoma brucei the function of Pam18 has been replaced by the non-orthologous euglenozoan-specific J-protein TbPam27. TbPam27 is specifically required for the import of mitochondrial presequence-containing but not for carrier proteins. Similar to yeast Pam18, TbPam27 requires an intact J-domain to function. Surprisingly, T. brucei still contains a bona fide Pam18 orthologue that, while essential for normal growth, is not involved in protein import. Thus, during evolution of kinetoplastids, Pam18 has been replaced by TbPam27. We propose that this replacement is linked to the transition from two ancestral and functionally distinct TIM complexes, found in most eukaryotes, to the single bifunctional TIM complex present in trypanosomes.

Neutral evolution of cellular phenotypes.

Eukaryotes exhibit a great diversity of cellular and subcellular morphologies, but their basic underlying architecture is fairly constant. All have a nucleus, Golgi, cytoskeleton, plasma membrane, vesicles, ribosomes, and all known lineages but one have mitochondrion-related organelles. Moreover, most eukaryotes undergo processes such as mitosis, meiosis, DNA recombination, and often perform feats such as phagocytosis, and amoeboid and flagellar movement. With all of these commonalities, it is obvious that eukaryotes evolved from a common ancestor, but it is not obvious how eukaryotes came to have their diverse structural phenotypes. Are these phenotypes adaptations to particular niches, their evolution dominated by positive natural selection? Or is eukaryotic cellular diversity substantially the product of neutral evolutionary processes, with adaptation either illusory or a secondary consequence? In this paper, we outline how a hierarchical view of phenotype can be used to articulate a neutral theory of phenotypic evolution, involving processes such as gene loss, gene replacement by homologues or analogues, gene duplication followed by subfunctionalization, and constructive neutral evolution. We suggest that neutral iterations of these processes followed by entrenchment of their products can explain much of the diversity of cellular, developmental, and biochemical phenotypes of unicellular eukaryotes and should be explored in addition to adaptive explanations.

Nephromyces Represents a Diverse and Novel Lineage of the Apicomplexa That Has Retained Apicoplasts.

A most interesting exception within the parasitic Apicomplexa is Nephromyces, an extracellular, probably mutualistic, endosymbiont found living inside molgulid ascidian tunicates (i.e., sea squirts). Even though Nephromyces is now known to be an apicomplexan, many other questions about its nature remain unanswered. To gain further insights into the biology and evolutionary history of this unusual apicomplexan, we aimed to 1) find the precise phylogenetic position of Nephromyces within the Apicomplexa, 2) search for the apicoplast genome of Nephromyces, and 3) infer the major metabolic pathways in the apicoplast of Nephromyces. To do this, we sequenced a metagenome and a metatranscriptome from the molgulid renal sac, the specialized habitat where Nephromyces thrives. Our phylogenetic analyses of conserved nucleus-encoded genes robustly suggest that Nephromyces is a novel lineage sister to the Hematozoa, which comprises both the Haemosporidia (e.g., Plasmodium) and the Piroplasmida (e.g., Babesia and Theileria). Furthermore, a survey of the renal sac metagenome revealed 13 small contigs that closely resemble the genomes of the nonphotosynthetic reduced plastids, or apicoplasts, of other apicomplexans. We show that these apicoplast genomes correspond to a diverse set of most closely related but genetically divergent Nephromyces lineages that co-inhabit a single tunicate host. In addition, the apicoplast of Nephromyces appears to have retained all biosynthetic pathways inferred to have been ancestral to parasitic apicomplexans. Our results shed light on the evolutionary history of the only probably mutualistic apicomplexan known, Nephromyces, and provide context for a better understanding of its life style and intricate symbiosis.

An updated phylogeny of the Alphaproteobacteria reveals that the parasitic Rickettsiales and Holosporales have independent origins.

The Alphaproteobacteria is an extraordinarily diverse and ancient group of bacteria. Previous attempts to infer its deep phylogeny have been plagued with methodological artefacts. To overcome this, we analyzed a dataset of 200 single-copy and conserved genes and employed diverse strategies to reduce compositional artefacts. Such strategies include using novel dataset-specific profile mixture models and recoding schemes, and removing sites, genes and taxa that are compositionally biased. We show that the Rickettsiales and Holosporales (both groups of intracellular parasites of eukaryotes) are not sisters to each other, but instead, the Holosporales has a derived position within the Rhodospirillales. A synthesis of our results also leads to an updated proposal for the higher-level taxonomy of the Alphaproteobacteria. Our robust consensus phylogeny will serve as a framework for future studies that aim to place mitochondria, and novel environmental diversity, within the Alphaproteobacteria.

Cell Biology: Functional Conservation, Structural Divergence, and Surprising Convergence in the MICOS Complex of Trypanosomes.

The MICOS complex is conserved across eukaryotes, but little is known about it outside the group that comprises animals and fungi. A new study finds that mitochondria of trypanosomatid parasites bear a divergent MICOS with both ancestral and derived subunits, but with conserved functions in crista development and membrane contact-site formation.

Microbial eukaryotes have adapted to hypoxia by horizontal acquisitions of a gene involved in rhodoquinone biosynthesis.

Under hypoxic conditions, some organisms use an electron transport chain consisting of only complex I and II (CII) to generate the proton gradient essential for ATP production. In these cases, CII functions as a fumarate reductase that accepts electrons from a low electron potential quinol, rhodoquinol (RQ). To clarify the origins of RQ-mediated fumarate reduction in eukaryotes, we investigated the origin and function of rquA, a gene encoding an RQ biosynthetic enzyme. RquA is very patchily distributed across eukaryotes and bacteria adapted to hypoxia. Phylogenetic analyses suggest lateral gene transfer (LGT) of rquA from bacteria to eukaryotes occurred at least twice and the gene was transferred multiple times amongst protists. We demonstrate that RquA functions in the mitochondrion-related organelles of the anaerobic protist Pygsuia and is correlated with the presence of RQ. These analyses reveal the role of gene transfer in the evolutionary remodeling of mitochondria in adaptation to hypoxia.

The Origin and Diversification of Mitochondria.

Mitochondria are best known for their role in the generation of ATP by aerobic respiration. Yet, research in the past half century has shown that they perform a much larger suite of functions and that these functions can vary substantially among diverse eukaryotic lineages. Despite this diversity, all mitochondria derive from a common ancestral organelle that originated from the integration of an endosymbiotic alphaproteobacterium into a host cell related to Asgard Archaea. The transition from endosymbiotic bacterium to permanent organelle entailed a massive number of evolutionary changes including the origins of hundreds of new genes and a protein import system, insertion of membrane transporters, integration of metabolism and reproduction, genome reduction, endosymbiotic gene transfer, lateral gene transfer and the retargeting of proteins. These changes occurred incrementally as the endosymbiont and the host became integrated. Although many insights into this transition have been gained, controversy persists regarding the nature of the original endosymbiont, its initial interactions with the host and the timing of its integration relative to the origin of other features of eukaryote cells. Since the establishment of the organelle, proteins have been gained, lost, transferred and retargeted as mitochondria have specialized into the spectrum of functional types seen across the eukaryotic tree of life.