Cytoplasmic Polyadenylation Is an Ancestral Hallmark of Early Development in Animals.

Differential regulation of gene expression has produced the astonishing diversity of life on Earth. Understanding the origin and evolution of mechanistic innovations for control of gene expression is therefore integral to evolutionary and developmental biology. Cytoplasmic polyadenylation is the biochemical extension of polyadenosine at the 3'-end of cytoplasmic mRNAs. This process regulates the translation of specific maternal transcripts and is mediated by the Cytoplasmic Polyadenylation Element-Binding Protein family (CPEBs). Genes that code for CPEBs are amongst a very few that are present in animals but missing in nonanimal lineages. Whether cytoplasmic polyadenylation is present in non-bilaterian animals (i.e., sponges, ctenophores, placozoans, and cnidarians) remains unknown. We have conducted phylogenetic analyses of CPEBs, and our results show that CPEB1 and CPEB2 subfamilies originated in the animal stem lineage. Our assessment of expression in the sea anemone, Nematostella vectensis (Cnidaria), and the comb jelly, Mnemiopsis leidyi (Ctenophora), demonstrates that maternal expression of CPEB1 and the catalytic subunit of the cytoplasmic polyadenylation machinery (GLD2) is an ancient feature that is conserved across animals. Furthermore, our measurements of poly(A)-tail elongation reveal that key targets of cytoplasmic polyadenylation are shared between vertebrates, cnidarians, and ctenophores, indicating that this mechanism orchestrates a regulatory network that is conserved throughout animal evolution. We postulate that cytoplasmic polyadenylation through CPEBs was a fundamental innovation that contributed to animal evolution from unicellular life.

Evolutionary origin of the nervous system from Ctenophora prospective.

Nervous system is one of the key adaptations underlying the evolutionary success of the majority of animal groups. Ctenophores (or comb jellies) are gelatinous marine invertebrates that were probably the first lineage to diverge from the rest of animals. Due to the key phylogenetic position and multiple unique adaptations, the noncentralized nervous system of comb jellies has been in the center of the debate around the origin of the nervous system in the animal kingdom and whether it happened only once or twice. Here, we discuss the latest findings in ctenophore neuroscience and multiple challenges on the way to build a clear evolutionary picture of the origin of the nervous system.

Light sensitivity in Beroidae ctenophores: Insights from laboratory studies and genomics.

Light detection underlies a variety of animal behaviors, including those related to spatial orientation, feeding, avoidance of predators, and reproduction. Ctenophores are likely the oldest animal group in which light sensitivity based on opsins evolved, so they may still have the ancestral molecular mechanisms for photoreception. However, knowledge about ctenophore photosensitivity, associated morphological structures, molecular mechanisms involved, and behavioral reactions is limited and fragmented. We present the initial experiments on the responses of adult Beroe ovata to high-intensity light exposure with different spectra and photosensitivity in various parts of the animal's body. Ctenophores have shown a consistent behavioral response when their aboral organ is exposed to a household-grade laser in the violet spectrum. To investigate the genes responsible for the photosensitivity of Beroidae, we have analyzed transcriptome and genome-wide datasets. We identified three opsins in Beroe that are homologous to those found in Mnemiopsis leidyi (Lobata) and Pleurobrachia bachei (Cydippida). These opsins form clades Ctenopsin1, 2, and 3, respectively. Ctenopsin3 is significantly distinct from other ctenophore opsins and clustered outside the main animal opsin groups. The Ctenopsin1 and Ctenopsin2 groups are sister clusters within the canonical animal opsin tree. These two groups could have originated from gene duplication in the common ancestor of the species we studied and then developed independently in different lineages of Ctenophores. So far, there is no evidence of additional expansion of the opsin family in ctenophore evolution. The involvement of ctenophore opsins in photoreception is discussed by analyzing their protein structures.

Chemical cognition: chemoconnectomics and convergent evolution of integrative systems in animals.

Neurons underpin cognition in animals. However, the roots of animal cognition are elusive from both mechanistic and evolutionary standpoints. Two conceptual frameworks both highlight and promise to address these challenges. First, we discuss evidence that animal neural and other integrative systems evolved more than once (convergent evolution) within basal metazoan lineages, giving us unique experiments by Nature for future studies. The most remarkable examples are neural systems in ctenophores and neuroid-like systems in placozoans and sponges. Second, in addition to classical synaptic wiring, a chemical connectome mediated by hundreds of signal molecules operates in tandem with neurons and is the most information-rich source of emerging properties and adaptability. The major gap-dynamic, multifunctional chemical micro-environments in nervous systems-is not understood well. Thus, novel tools and information are needed to establish mechanistic links between orchestrated, yet cell-specific, volume transmission and behaviors. Uniting what we call chemoconnectomics and analyses of the cellular bases of behavior in basal metazoan lineages arguably would form the foundation for deciphering the origins and early evolution of elementary cognition and intelligence.

[Spatial Reconstruction of TRPC Mechanoreceptors of the Ctenophore Mnemiopsis leidyi A. Agassiz, 1865].

The ctenophore Mnemiopsis leidyi A. Agassiz, 1865 responds to gentle mechanical stimulus with intense luminescence; however, the mechanism of this phenomenon is unknown. We searched for possible mechanosensitive receptors that initiate signal transduction resulting in photoprotein luminescence. The three orthologous genes of mouse (5z96) and drosophila (5vkq) TRPC-proteins, such as ML234550a-PA (860 a.a.), ML03701a-PA (828 a.a.), and ML038011a-PA (1395 a.a.), were found in the M. leidyi genome. The latter protein contains a long ankyrin helix consisting of 16 ANK domains. Study of the annotated domains and the network of interactions between the interactome proteins suggests that the ML234550a-PA and ML03701a-PA proteins carry out cytoplasmic transduction, but ML038011a-PA provides intranuclear transduction of mechanical signals. Spatial reconstruction of the studied proteins revealed differences in their structure, which may be related to various functions of these proteins in the cell. The question of which of these proteins is involved in the initiation of luminescence after mechanical stimulation is discussed.

Rooting the Animal Tree of Life.

Identifying our most distant animal relatives has emerged as one of the most challenging problems in phylogenetics. This debate has major implications for our understanding of the origin of multicellular animals and of the earliest events in animal evolution, including the origin of the nervous system. Some analyses identify sponges as our most distant animal relatives (Porifera-sister hypothesis), and others identify comb jellies (Ctenophora-sister hypothesis). These analyses vary in many respects, making it difficult to interpret previous tests of these hypotheses. To gain insight into why different studies yield different results, an important next step in the ongoing debate, we systematically test these hypotheses by synthesizing 15 previous phylogenomic studies and performing new standardized analyses under consistent conditions with additional models. We find that Ctenophora-sister is recovered across the full range of examined conditions, and Porifera-sister is recovered in some analyses under narrow conditions when most outgroups are excluded and site-heterogeneous CAT models are used. We additionally find that the number of categories in site-heterogeneous models is sufficient to explain the Porifera-sister results. Furthermore, our cross-validation analyses show CAT models that recover Porifera-sister have hundreds of additional categories and fail to fit significantly better than site-heterogenuous models with far fewer categories. Systematic and standardized testing of diverse phylogenetic models suggests that we should be skeptical of Porifera-sister results both because they are recovered under such narrow conditions and because the models in these conditions fit the data no better than other models that recover Ctenophora-sister.

Microchemical identification of enantiomers in early-branching animals: Lineage-specific diversification in the usage of D-glutamate and D-aspartate.

D-amino acids are unique and essential signaling molecules in neural, hormonal, and immune systems. However, the presence of D-amino acids and their recruitment in early animals is mostly unknown due to limited information about prebilaterian metazoans. Here, we performed the comparative survey of L-/D-aspartate and L-/D-glutamate in representatives of four phyla of early-branching Metazoa: cnidarians (Aglantha); placozoans (Trichoplax), sponges (Sycon) and ctenophores (Pleurobrachia, Mnemiopsis, Bolinopsis, and Beroe), which are descendants of ancestral animal lineages distinct from Bilateria. Specifically, we used high-performance capillary electrophoresis for microchemical assays and quantification of the enantiomers. L-glutamate and L-aspartate were abundant analytes in all species studied. However, we showed that the placozoans, cnidarians, and sponges had high micromolar concentrations of D-aspartate, whereas D-glutamate was not detectable in our assays. In contrast, we found that in ctenophores, D-glutamate was the dominant enantiomer with no or trace amounts of D-aspartate. This situation illuminates prominent lineage-specific diversifications in the recruitment of D-amino acids and suggests distinct signaling functions of these molecules early in the animal evolution. We also hypothesize that a deep ancestry of such recruitment events might provide some constraints underlying the evolution of neural and other signaling systems in Metazoa.

The first identification of complete Eph-ephrin signalling in ctenophores and sponges reveals a role for neofunctionalization in the emergence of signalling domains.

BACKGROUND: Animals have a greater diversity of signalling pathways than their unicellular relatives, consistent with the evolution and expansion of these pathways occurring in parallel with the origin of animal multicellularity. However, the genomes of sponges and ctenophores - non-bilaterian basal animals - typically encode no, or far fewer, recognisable signalling ligands compared to bilaterians and cnidarians. For instance, the largest subclass of receptor tyrosine kinases (RTKs) in bilaterians, the Eph receptors (Ephs), are present in sponges and ctenophores, but their cognate ligands, the ephrins, have not yet been detected. RESULTS: Here, we use an iterative HMM analysis to identify for the first time membrane-bound ephrins in sponges and ctenophores. We also expand the number of Eph-receptor subtypes identified in these animals and in cnidarians. Both sequence and structural analyses are consistent with the Eph ligand binding domain (LBD) and the ephrin receptor binding domain (RBD) having evolved via the co-option of ancient galactose-binding (discoidin-domain)-like and monodomain cupredoxin domains, respectively. Although we did not detect a complete Eph-ephrin signalling pathway in closely-related unicellular holozoans or in other non-metazoan eukaryotes, truncated proteins with Eph receptor LBDs and ephrin RBDs are present in some choanoflagellates. Together, these results indicate that Eph-ephrin signalling was present in the last common ancestor of extant metazoans, and perhaps even in the last common ancestor of animals and choanoflagellates. Either scenario pushes the origin of Eph-ephrin signalling back much earlier than previously reported. CONCLUSIONS: We propose that the Eph-LBD and ephrin-RBD, which were ancestrally localised in the cytosol, became linked to the extracellular parts of two cell surface proteins before the divergence of sponges and ctenophores from the rest of the animal kingdom. The ephrin-RBD lost the ancestral capacity to bind copper, and the Eph-LBD became linked to an ancient RTK. The identification of divergent ephrin ligands in sponges and ctenophores suggests that these ligands evolve faster than their cognate receptors. As this may be a general phenomena, we propose that the sequence-structure approach used in this study may be usefully applied to other signalling systems where no, or a small number of, ligands have been identified.

A non-bilaterian perspective on the development and evolution of animal digestive systems.

Digestive systems and extracellular digestion are key animal features, but their emergence during early animal evolution is currently poorly understood. As the last common ancestor of non-bilaterian animal groups (sponges, ctenophores, placozoans and cnidarians) dates back to the beginning of animal life, their study and comparison provides important insights into the early evolution of digestive systems and functions. Here, I have compiled an overview of the development and cell biology of digestive tissues in non-bilaterian animals. I will highlight the fundamental differences between extracellular and intracellular digestive processes, and how these are distributed among animals. Cnidarians (e.g. sea anemones, corals, jellyfish), the phylogenetic outgroup of bilaterians (e.g. vertebrates, flies, annelids), occupy a key position to reconstruct the evolution of bilaterian gut evolution. A major focus will therefore lie on the development and cell biology of digestive tissues in cnidarians, especially sea anemones, and how they compare to bilaterian gut tissues. In that context, I will also review how a recent study on the gastrula fate map of the sea anemone Nematostella vectensis challenges our long-standing conceptions on the evolution of cnidarian and bilaterian germ layers and guts.

Jellyfish-Associated Microbiome in the Marine Environment: Exploring Its Biotechnological Potential.

Despite accumulating evidence of the importance of the jellyfish-associated microbiome to jellyfish, its potential relevance to blue biotechnology has only recently been recognized. In this review, we emphasize the biotechnological potential of host⁻microorganism systems and focus on gelatinous zooplankton as a host for the microbiome with biotechnological potential. The basic characteristics of jellyfish-associated microbial communities, the mechanisms underlying the jellyfish-microbe relationship, and the role/function of the jellyfish-associated microbiome and its biotechnological potential are reviewed. It appears that the jellyfish-associated microbiome is discrete from the microbial community in the ambient seawater, exhibiting a certain degree of specialization with some preferences for specific jellyfish taxa and for specific jellyfish populations, life stages, and body parts. In addition, different sampling approaches and methodologies to study the phylogenetic diversity of the jellyfish-associated microbiome are described and discussed. Finally, some general conclusions are drawn from the existing literature and future research directions are highlighted on the jellyfish-associated microbiome.

A software tool 'CroCo' detects pervasive cross-species contamination in next generation sequencing data.

BACKGROUND: Multiple RNA samples are frequently processed together and often mixed before multiplex sequencing in the same sequencing run. While different samples can be separated post sequencing using sample barcodes, the possibility of cross contamination between biological samples from different species that have been processed or sequenced in parallel has the potential to be extremely deleterious for downstream analyses. RESULTS: We present CroCo, a software package for identifying and removing such cross contaminants from assembled transcriptomes. Using multiple, recently published sequence datasets, we show that cross contamination is consistently present at varying levels in real data. Using real and simulated data, we demonstrate that CroCo detects contaminants efficiently and correctly. Using a real example from a molecular phylogenetic dataset, we show that contaminants, if not eliminated, can have a decisive, deleterious impact on downstream comparative analyses. CONCLUSIONS: Cross contamination is pervasive in new and published datasets and, if undetected, can have serious deleterious effects on downstream analyses. CroCo is a database-independent, multi-platform tool, designed for ease of use, that efficiently and accurately detects and removes cross contamination in assembled transcriptomes to avoid these problems. We suggest that the use of CroCo should become a standard cleaning step when processing multiple samples for transcriptome sequencing.

Characterization of mitochondrial proteomes of nonbilaterian animals.

Mitochondria require ~1,500 proteins for their maintenance and proper functionality, which constitute the mitochondrial proteome (mt-proteome). Although a few of these proteins, mostly subunits of the electron transport chain complexes, are encoded in mitochondrial DNA (mtDNA), the vast majority are encoded in the nuclear genome and imported to the organelle. Previous studies have shown a continuous and complex evolution of mt-proteome among eukaryotes. However, there was less attention paid to mt-proteome evolution within Metazoa, presumably because animal mtDNA and, by extension, animal mitochondria are often considered to be uniform. In this analysis, two bioinformatic approaches (Orthologue-detection and Mitochondrial Targeting Sequence prediction) were used to identify mt-proteins in 23 species from four nonbilaterian phyla: Cnidaria, Ctenophora, Placozoa, and Porifera, as well as two choanoflagellates, the closest animal relatives. Our results revealed a large variation in mt-proteome in nonbilaterian animals in size and composition. Myxozoans, highly reduced cnidarian parasites, possessed the smallest inferred mitochondrial proteomes, while calcareous sponges possessed the largest. About 513 mitochondrial orthologous groups were present in all nonbilaterian phyla and human. Interestingly, 42 human mitochondrial proteins were not identified in any nonbilaterian species studied and represent putative innovations along the bilaterian branch. Several of these proteins were involved in apoptosis and innate immunity, two processes known to evolve within Metazoa. Conversely, several proteins identified as mitochondrial in nonbilaterian phyla and animal outgroups were absent in human, representing cases of possible loss. Finally, a few human cytosolic proteins, such as histones and cytosolic ribosomal proteins, were predicted to be targeted to mitochondria in nonbilaterian animals. Overall, our analysis provides the first step in characterization of mt-proteomes in nonbilaterian animals and understanding evolution of animal mt-proteome. © 2018 IUBMB Life, 70(12):1289-1301, 2018.

Genomic data do not support comb jellies as the sister group to all other animals.

Understanding how complex traits, such as epithelia, nervous systems, muscles, or guts, originated depends on a well-supported hypothesis about the phylogenetic relationships among major animal lineages. Traditionally, sponges (Porifera) have been interpreted as the sister group to the remaining animals, a hypothesis consistent with the conventional view that the last common animal ancestor was relatively simple and more complex body plans arose later in evolution. However, this premise has recently been challenged by analyses of the genomes of comb jellies (Ctenophora), which, instead, found ctenophores as the sister group to the remaining animals (the "Ctenophora-sister" hypothesis). Because ctenophores are morphologically complex predators with true epithelia, nervous systems, muscles, and guts, this scenario implies these traits were either present in the last common ancestor of all animals and were lost secondarily in sponges and placozoans (Trichoplax) or, alternatively, evolved convergently in comb jellies. Here, we analyze representative datasets from recent studies supporting Ctenophora-sister, including genome-scale alignments of concatenated protein sequences, as well as a genomic gene content dataset. We found no support for Ctenophora-sister and conclude it is an artifact resulting from inadequate methodology, especially the use of simplistic evolutionary models and inappropriate choice of species to root the metazoan tree. Our results reinforce a traditional scenario for the evolution of complexity in animals, and indicate that inferences about the evolution of Metazoa based on the Ctenophora-sister hypothesis are not supported by the currently available data.

Error, signal, and the placement of Ctenophora sister to all other animals.

Elucidating relationships among early animal lineages has been difficult, and recent phylogenomic analyses place Ctenophora sister to all other extant animals, contrary to the traditional view of Porifera as the earliest-branching animal lineage. To date, phylogenetic support for either ctenophores or sponges as sister to other animals has been limited and inconsistent among studies. Lack of agreement among phylogenomic analyses using different data and methods obscures how complex traits, such as epithelia, neurons, and muscles evolved. A consensus view of animal evolution will not be accepted until datasets and methods converge on a single hypothesis of early metazoan relationships and putative sources of systematic error (e.g., long-branch attraction, compositional bias, poor model choice) are assessed. Here, we investigate possible causes of systematic error by expanding taxon sampling with eight novel transcriptomes, strictly enforcing orthology inference criteria, and progressively examining potential causes of systematic error while using both maximum-likelihood with robust data partitioning and Bayesian inference with a site-heterogeneous model. We identified ribosomal protein genes as possessing a conflicting signal compared with other genes, which caused some past studies to infer ctenophores and cnidarians as sister. Importantly, biases resulting from elevated compositional heterogeneity or elevated substitution rates are ruled out. Placement of ctenophores as sister to all other animals, and sponge monophyly, are strongly supported under multiple analyses, herein.

The maternal-zygotic transition and zygotic activation of the Mnemiopsis leidyi genome occurs within the first three cleavage cycles.

The maternal-zygotic transition (MZT) describes the developmental reprogramming of gene expression marked by the degradation of maternally supplied gene products and activation of the zygotic genome. While the timing and duration of the MZT vary among taxa, little is known about early-stage transcriptional dynamics in the non-bilaterian phylum Ctenophora. We sought to better understand the extent of maternal mRNA loading and subsequent differential transcript abundance during the earliest stages of development by performing comprehensive RNA-sequencing-based analyses of mRNA abundance in single- and eight-cell stage embryos in the lobate ctenophore Mnemiopsis leidyi. We found 1,908 contigs with significant differential abundance between single- and eight-cell stages, of which 1,208 contigs were more abundant at the single-cell stage and 700 contigs were more abundant at the eight-cell stage. Of the differentially abundant contigs, 267 were exclusively present in the eight-cell samples, providing strong evidence that both the MZT and zygotic genome activation (ZGA) have commenced by the eight-cell stage. Many highly abundant transcripts encode genes involved in molecular mechanisms critical to the MZT, such as maternal transcript degradation, serine/threonine kinase activity, and chromatin remodeling. Our results suggest that chromosomal restructuring, which is critical to ZGA and the initiation of transcriptional regulation necessary for normal development, begins by the third cleavage within 1.5 hr post-fertilization in M. leidyi.

Retracing the path of planar cell polarity.

BACKGROUND: The Planar Cell Polarity pathway (PCP) has been described as the main feature involved in patterning cell orientation in bilaterian tissues. Recently, a similar phenomenon was revealed in cnidarians, in which the inhibition of this pathway results in the absence of cilia orientation in larvae, consequently proving the functional conservation of PCP signaling between Cnidaria and Bilateria. Nevertheless, despite the growing accumulation of databases concerning basal lineages of metazoans, very few information concerning the existence of PCP components have been gathered outside of Bilateria and Cnidaria. Thus, the origin of this module or its prevalence in early emerging metazoans has yet to be elucidated. RESULTS: The present study addresses this question by investigating the genomes and transcriptomes from all poriferan lineages in addition to Trichoplax (Placozoa) and Mnemiopsis (Ctenophora) genomes for the presence of the core components of this pathway. Our results confirm that several PCP components are metazoan innovations. In addition, we show that all members of the PCP pathway, including a bona fide Strabismus ortholog (Van gogh), are retrieved only in one sponge lineage (Homoscleromorpha) out of four. This highly suggests that the full PCP pathway dates back at least to the emergence of homoscleromorph sponges. Consequently, several secondary gene losses would have occurred in the three other poriferan lineages including Amphimedon queenslandica (Demospongiae). Several proteins were not retrieved either in placozoans or ctenophores leading us to discuss the difficulties to predict orthologous proteins in basally branching animals. Finally, we reveal how the study of multigene families may be helpful to unravel the relationships at the base of the metazoan tree. CONCLUSION: The PCP pathway antedates the radiation of Porifera and may have arisen in the last common ancestor of animals. Oscarella species now appear as key organisms to understand the ancestral function of PCP signaling and its potential links with Wnt pathways.

Convergent evolution of neural systems in ctenophores.

Neurons are defined as polarized secretory cells specializing in directional propagation of electrical signals leading to the release of extracellular messengers - features that enable them to transmit information, primarily chemical in nature, beyond their immediate neighbors without affecting all intervening cells en route. Multiple origins of neurons and synapses from different classes of ancestral secretory cells might have occurred more than once during ~600 million years of animal evolution with independent events of nervous system centralization from a common bilaterian/cnidarian ancestor without the bona fide central nervous system. Ctenophores, or comb jellies, represent an example of extensive parallel evolution in neural systems. First, recent genome analyses place ctenophores as a sister group to other animals. Second, ctenophores have a smaller complement of pan-animal genes controlling canonical neurogenic, synaptic, muscle and immune systems, and developmental pathways than most other metazoans. However, comb jellies are carnivorous marine animals with a complex neuromuscular organization and sophisticated patterns of behavior. To sustain these functions, they have evolved a number of unique molecular innovations supporting the hypothesis of massive homoplasies in the organization of integrative and locomotory systems. Third, many bilaterian/cnidarian neuron-specific genes and 'classical' neurotransmitter pathways are either absent or, if present, not expressed in ctenophore neurons (e.g. the bilaterian/cnidarian neurotransmitter, γ-amino butyric acid or GABA, is localized in muscles and presumed bilaterian neuron-specific RNA-binding protein Elav is found in non-neuronal cells). Finally, metabolomic and pharmacological data failed to detect either the presence or any physiological action of serotonin, dopamine, noradrenaline, adrenaline, octopamine, acetylcholine or histamine - consistent with the hypothesis that ctenophore neural systems evolved independently from those in other animals. Glutamate and a diverse range of secretory peptides are first candidates for ctenophore neurotransmitters. Nevertheless, it is expected that other classes of signal and neurogenic molecules would be discovered in ctenophores as the next step to decipher one of the most distinct types of neural organization in the animal kingdom.

The ctenophore lineage is older than sponges? That cannot be right! Or can it?

Recent phylogenetic analyses resulting from collection of whole genome data suggest that ctenophores, or comb jellies, are sister to all other animals. Even before publication, this result prompted discussion among researchers. Here, I counter common criticisms raised about this result and show that assumptions placing sponges as the basal-most extant animal lineage are based on limited evidence and questionable premises. For example, the idea that sponges are simple and the reported similarity of sponge choanocytes to Choanflagellata do not provide useful characters for determining the positions of sponges within the animal tree. Intertwined with discussion of basal metazoan phylogeny is consideration of the evolution of neuronal systems. Recent data show that neural systems of ctenophores are vastly different from those of other animals and use different sets of cellular and genetic mechanisms. Thus, neural systems appear to have at least two independent origins regardless of whether ctenophores or sponges are the earliest branching extant animal lineage.

Dynamic evolution of mitochondrial ribosomal proteins in Holozoa.

We studied the highly dynamic evolution of mitochondrial ribosomal proteins (MRPs) in Holozoa. Most major clades within Holozoa are characterized by gains and/or losses of MRPs. The usefulness of gains of MRPs as rare genomic changes in phylogenetics is undermined by the high frequency of secondary losses. However, phylogenetic analyses of the MRP sequences provide evidence for the Acrosomata hypothesis, a sister group relationship between Ctenophora and Bilateria. An extensive restructuring of the mitochondrial genome and, as a consequence, of the mitochondrial ribosomes occurred in the ancestor of metazoans. The last MRP genes encoded in the mitochondrial genome were either moved to the nuclear genome or were lost. The strong decrease in size of the mitochondrial genome was probably caused by selection for rapid replication of mitochondrial DNA during oogenesis in the metazoan ancestor. A phylogenetic analysis of MRPL56 sequences provided evidence for a horizontal gene transfer of the corresponding MRP gene between metazoans and Dictyostelidae (Amoebozoa). The hypothesis that the requisition of additional MRPs compensated for a loss of rRNA segments in the mitochondrial ribosomes is corroborated by a significant negative correlation between the number of MRPs and length of the rRNA. Newly acquired MRPs evolved faster than bacterial MRPs and positions in eukaryote-specific MRPs were more strongly affected by coevolution than positions in prokaryotic MRPs in accordance with the necessity to fit these proteins into the pre-existing structure of the mitoribosome.