Massive colonization of protein-coding exons by selfish genetic elements in Paramecium germline genomes.

Ciliates are unicellular eukaryotes with both a germline genome and a somatic genome in the same cytoplasm. The somatic macronucleus (MAC), responsible for gene expression, is not sexually transmitted but develops from a copy of the germline micronucleus (MIC) at each sexual generation. In the MIC genome of Paramecium tetraurelia, genes are interrupted by tens of thousands of unique intervening sequences called internal eliminated sequences (IESs), which have to be precisely excised during the development of the new MAC to restore functional genes. To understand the evolutionary origin of this peculiar genomic architecture, we sequenced the MIC genomes of 9 Paramecium species (from approximately 100 Mb in Paramecium aurelia species to >1.5 Gb in Paramecium caudatum). We detected several waves of IES gains, both in ancestral and in more recent lineages. While the vast majority of IESs are single copy in present-day genomes, we identified several families of mobile IESs, including nonautonomous elements acquired via horizontal transfer, which generated tens to thousands of new copies. These observations provide the first direct evidence that transposable elements can account for the massive proliferation of IESs in Paramecium. The comparison of IESs of different evolutionary ages indicates that, over time, IESs shorten and diverge rapidly in sequence while they acquire features that allow them to be more efficiently excised. We nevertheless identified rare cases of IESs that are under strong purifying selection across the aurelia clade. The cases examined contain or overlap cellular genes that are inactivated by excision during development, suggesting conserved regulatory mechanisms. Similar to the evolution of introns in eukaryotes, the evolution of Paramecium IESs highlights the major role played by selfish genetic elements in shaping the complexity of genome architecture and gene expression.

The Role of Homology and Orthology in the Phylogenomic Analysis of Metazoan Gene Content.

Resolving the relationships of animals (Metazoa) is crucial to our understanding of the origin of key traits such as muscles, guts, and nerves. However, a broadly accepted metazoan consensus phylogeny has yet to emerge. In part, this is because the genomes of deeply diverging and fast-evolving lineages may undergo significant gene turnover, reducing the number of orthologs shared with related phyla. This can limit the usefulness of traditional phylogenetic methods that rely on alignments of orthologous sequences. Phylogenetic analysis of gene content has the potential to circumvent this orthology requirement, with binary presence/absence of homologous gene families representing a source of phylogenetically informative characters. Applying binary substitution models to the gene content of 26 complete animal genomes, we demonstrate that patterns of gene conservation differ markedly depending on whether gene families are defined by orthology or homology, that is, whether paralogs are excluded or included. We conclude that the placement of some deeply diverging lineages may exceed the limit of resolution afforded by the current methods based on comparisons of orthologous protein sequences, and novel approaches are required to fully capture the evolutionary signal from genes within genomes.

Ancient crested penguin constrains timing of recruitment into seabird hotspot.

New Zealand is a globally significant hotspot for seabird diversity, but the sparse fossil record for most seabird lineages has impeded our understanding of how and when this hotspot developed. Here, we describe multiple exceptionally well-preserved specimens of a new species of penguin from tightly dated (3.36-3.06 Ma) Pliocene deposits in New Zealand. Bayesian and parsimony analyses place Eudyptes atatu sp. nov. as the sister species to all extant and recently extinct members of the crested penguin genus Eudyptes. The new species has a markedly more slender upper beak and mandible compared with other Eudyptes penguins. Our combined evidence approach reveals that deep bills evolved in both crested and stiff-tailed penguins (Pygoscelis) during the Pliocene. That deep bills arose so late in the greater than 60 million year evolutionary history of penguins suggests that dietary shifts may have occurred as wind-driven Pliocene upwelling radically restructured southern ocean ecosystems. Ancestral area reconstructions using BioGeoBEARS identify New Zealand as the most likely ancestral area for total-group penguins, crown penguins and crested penguins. Our analyses provide a timeframe for recruitment of crown penguins into the New Zealand avifauna, indicating this process began in the late Neogene and was completed via multiple waves of colonizing lineages.

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.

Mitochondrial DNA of Clathrina clathrus (Calcarea, Calcinea): six linear chromosomes, fragmented rRNAs, tRNA editing, and a novel genetic code.

Sponges (phylum Porifera) are a large and ancient group of morphologically simple but ecologically important aquatic animals. Although their body plan and lifestyle are relatively uniform, sponges show extensive molecular and genetic diversity. In particular, mitochondrial genomes from three of the four previously studied classes of Porifera (Demospongiae, Hexactinellida, and Homoscleromorpha) have distinct gene contents, genome organizations, and evolutionary rates. Here, we report the mitochondrial genome of Clathrina clathrus (Calcinea, Clathrinidae), a representative of the fourth poriferan class, the Calcarea, which proves to be the most unusual. Clathrina clathrus mitochondrial DNA (mtDNA) consists of six linear chromosomes 7.6-9.4 kb in size and encodes at least 37 genes: 13 protein codings, 2 ribosomal RNAs (rRNAs), and 24 transfer RNAs (tRNAs). Protein genes include atp9, which has now been found in all major sponge lineages, but no atp8. Our analyses further reveal the presence of a novel genetic code that involves unique reassignments of the UAG codons from termination to tyrosine and of the CGN codons from arginine to glycine. Clathrina clathrus mitochondrial rRNAs are encoded in three (srRNA) and ≥6 (lrRNA) fragments distributed out of order and on several chromosomes. The encoded tRNAs contain multiple mismatches in the aminoacyl acceptor stems that are repaired posttranscriptionally by 3'-end RNA editing. Although our analysis does not resolve the phylogenetic position of calcareous sponges, likely due to their high rates of mitochondrial sequence evolution, it confirms mtDNA as a promising marker for population studies in this group. The combination of unusual mitochondrial features in C. clathrus redefines the extremes of mtDNA evolution in animals and further argues against the idea of a "typical animal mtDNA."

Extensive and evolutionarily persistent mitochondrial tRNA editing in Velvet Worms (phylum Onychophora).

Mitochondrial genomes of onychophorans (velvet worms) present an interesting problem: Some previous studies reported them lacking several transfer RNA (tRNA) genes, whereas others found that all their tRNA genes were present but severely reduced. To resolve this discrepancy, we determined complete mitochondrial DNA (mtDNA) sequences of the onychophorans Oroperipatus sp. and Peripatoides sympatrica as well as cDNA sequences from 14 and 10 of their tRNAs, respectively. We show that tRNA genes in these genomes are indeed highly reduced and encode truncated molecules, which are restored to more conventional structures by extensive tRNA editing. During this editing process, up to 34 nucleotides are added to the tRNA sequences encoded in Oroperipatus sp. mtDNA, rebuilding the aminoacyl acceptor stem, the TΨC arm, and in some extreme cases, the variable arm and even a part of the anticodon stem. The editing is less extreme in P. sympatrica in which at least a part of the TΨC arm is always encoded in mtDNA. When the entire TΨC arm is added de novo in Oroperipatus sp., the sequence of this arm is either identical or similar among different tRNA species, yet the sequences show substantial variation for each tRNA. These observations suggest that the arm is rebuilt, at least in part, by a template-independent mechanism and argue against the alternative possibility that tRNA genes or their parts are imported from the nucleus. By contrast, the 3' end of the aminoacyl acceptor stem is likely restored by a template-dependent mechanism. The extreme tRNA editing reported here has been preserved for >140 My as it was found in both extant families of onychophorans. Furthermore, a similar type of tRNA editing may be present in several other groups of arthropods, which show a high degree of tRNA gene reduction in their mtDNA.

Parallel loss of nuclear-encoded mitochondrial aminoacyl-tRNA synthetases and mtDNA-encoded tRNAs in Cnidaria.

Unlike most animal mitochondrial (mt) genomes, which encode a set of 22 transfer RNAs (tRNAs) sufficient for mt protein synthesis, those of cnidarians have only retained one or two tRNA genes. Whether the missing cnidarian mt-tRNA genes relocated outside the main mt chromosome or were lost remains unclear. It is also unknown what impact the loss of tRNA genes had on other components of the mt translational machinery. Here, we explored the nuclear genome of the cnidarian Nematostella vectensis for the presence of mt-tRNA genes and their corresponding mt aminoacyl-tRNA synthetases (mt-aaRS). We detected no candidates for mt-tRNA genes and only two mt-aaRS orthologs. At the same time, we found that all but one cytosolic aaRS appear to be targeted to mitochondria. These results indicate that the loss of mt-tRNAs in Cnidaria is genuine and occurred in parallel with the loss of nuclear-encoded mt-aaRS. Our phylogenetic analyses of individual aaRS revealed that although the nearly total loss of mt-aaRS is rare, aaRS gene deletion and replacement have occurred throughout the evolution of Metazoa.

Well-Annotated microRNAomes Do Not Evidence Pervasive miRNA Loss.

microRNAs are conserved noncoding regulatory factors implicated in diverse physiological and developmental processes in multicellular organisms, as causal macroevolutionary agents and for phylogeny inference. However, the conservation and phylogenetic utility of microRNAs has been questioned on evidence of pervasive loss. Here, we show that apparent widespread losses are, largely, an artefact of poorly sampled and annotated microRNAomes. Using a curated data set of animal microRNAomes, we reject the view that miRNA families are never lost, but they are rarely lost (92% are never lost). A small number of families account for a majority of losses (1.7% of families account for >45% losses), and losses are associated with lineages exhibiting phenotypic simplification. Phylogenetic analyses based on the presence/absence of microRNA families among animal lineages, and based on microRNA sequences among Osteichthyes, demonstrate the power of these small data sets in phylogenetic inference. Perceptions of widespread evolutionary loss of microRNA families are due to the uncritical use of public archives corrupted by spurious microRNA annotations, and failure to discriminate false absences that occur because of incomplete microRNAome annotation.

Improved Modeling of Compositional Heterogeneity Supports Sponges as Sister to All Other Animals.

The relationships at the root of the animal tree have proven difficult to resolve, with the current debate focusing on whether sponges (phylum Porifera) or comb jellies (phylum Ctenophora) are the sister group of all other animals [1-5]. The choice of evolutionary models seems to be at the core of the problem because Porifera tends to emerge as the sister group of all other animals ("Porifera-sister") when site-specific amino acid differences are modeled (e.g., [6, 7]), whereas Ctenophora emerges as the sister group of all other animals ("Ctenophora-sister") when they are ignored (e.g., [8-11]). We show that two key phylogenomic datasets that previously supported Ctenophora-sister [10, 12] display strong heterogeneity in amino acid composition across sites and taxa and that no routinely used evolutionary model can adequately describe both forms of heterogeneity. We show that data-recoding methods [13-15] reduce compositional heterogeneity in these datasets and that models accommodating site-specific amino acid preferences can better describe the recoded datasets. Increased model adequacy is associated with significant topological changes in support of Porifera-sister. Because adequate modeling of the evolutionary process that generated the data is fundamental to recovering an accurate phylogeny [16-20], our results strongly support sponges as the sister group of all other animals and provide further evidence that Ctenophora-sister represents a tree reconstruction artifact. VIDEO ABSTRACT.

Animal Mitochondrial DNA as We Do Not Know It: mt-Genome Organization and Evolution in Nonbilaterian Lineages.

Animal mitochondrial DNA (mtDNA) is commonly described as a small, circular molecule that is conserved in size, gene content, and organization. Data collected in the last decade have challenged this view by revealing considerable diversity in animal mitochondrial genome organization. Much of this diversity has been found in nonbilaterian animals (phyla Cnidaria, Ctenophora, Placozoa, and Porifera), which, from a phylogenetic perspective, form the main branches of the animal tree along with Bilateria. Within these groups, mt-genomes are characterized by varying numbers of both linear and circular chromosomes, extra genes (e.g. atp9, polB, tatC), large variation in the number of encoded mitochondrial transfer RNAs (tRNAs) (0-25), at least seven different genetic codes, presence/absence of introns, tRNA and mRNA editing, fragmented ribosomal RNA genes, translational frameshifting, highly variable substitution rates, and a large range of genome sizes. This newly discovered diversity allows a better understanding of the evolutionary plasticity and conservation of animal mtDNA and provides insights into the molecular and evolutionary mechanisms shaping mitochondrial genomes.

Cytonuclear Interactions in the Evolution of Animal Mitochondrial tRNA Metabolism.

The evolution of mitochondrial information processing pathways, including replication, transcription and translation, is characterized by the gradual replacement of mitochondrial-encoded proteins with nuclear-encoded counterparts of diverse evolutionary origins. Although the ancestral enzymes involved in mitochondrial transcription and replication have been replaced early in eukaryotic evolution, mitochondrial translation is still carried out by an apparatus largely inherited from the α-proteobacterial ancestor. However, variation in the complement of mitochondrial-encoded molecules involved in translation, including transfer RNAs (tRNAs), provides evidence for the ongoing evolution of mitochondrial protein synthesis. Here, we investigate the evolution of the mitochondrial translational machinery using recent genomic and transcriptomic data from animals that have experienced the loss of mt-tRNAs, including phyla Cnidaria and Ctenophora, as well as some representatives of all four classes of Porifera. We focus on four sets of mitochondrial enzymes that directly interact with tRNAs: Aminoacyl-tRNA synthetases, glutamyl-tRNA amidotransferase, tRNA(Ile) lysidine synthetase, and RNase P. Our results support the observation that the fate of nuclear-encoded mitochondrial proteins is influenced by the evolution of molecules encoded in mitochondrial DNA, but in a more complex manner than appreciated previously. The data also suggest that relaxed selection on mitochondrial translation rather than coevolution between mitochondrial and nuclear subunits is responsible for elevated rates of evolution in mitochondrial translational proteins.

Eight new mtDNA sequences of glass sponges reveal an extensive usage of +1 frameshifting in mitochondrial translation.

Three previously studied mitochondrial genomes of glass sponges (phylum Porifera, class Hexactinellida) contained single nucleotide insertions in protein coding genes inferred as sites of +1 translational frameshifting. To investigate the distribution and evolution of these sites and to help elucidate the mechanism of frameshifting, we determined eight new complete or nearly complete mtDNA sequences from glass sponges and examined individual mitochondrial genes from three others. We found nine new instances of single nucleotide insertions in these sequences and analyzed them both comparatively and phylogenetically. The base insertions appear to have been gained and lost repeatedly in hexactinellid mt protein genes, suggesting no functional significance for the frameshifting sites. A high degree of sequence conservation, the presence of unusual tRNAs, and a distinct pattern of codon usage suggest the "out-of-frame pairing" model of translational frameshifting. Additionally, we provide evidence that relaxed selection pressure on glass sponge mtDNA - possibly a result of their low growth rates and deep-water lifestyle - has allowed frameshift insertions to be tolerated for hundreds of millions of years. Our study provides the first example of a phylogenetically diverse and extensive usage of translational frameshifting in animal mitochondrial coding sequences.

The twin-arginine subunit C in Oscarella: origin, evolution, and potential functional significance.

The twin-arginine translocation (Tat) pathway is a protein transport system that moves completely folded proteins across lipid membranes. Genes encoding components of the pathway have been found in the genomes of many Bacteria, Archaea, and eukaryotic organelles including chloroplasts, plant mitochondria, and the mitochondria of many protists. However, with a single exception, Tat genes are absent from the mitochondrial genomes of all animals. The only exception comes from the homoscleromorph sponges in the family Oscarellidae, whose mitochondrial genomes encode a gene for tatC, the largest subunit of the complex. Here, we explore the origin and evolution of the mitochondrial tatC gene in Oscarellidae, and use bioinformatic approaches to evaluate its functional significance. We conclude that tatC in Homoscleromorpha sponges was likely inherited from the ancestral proto-mitochondrial genome, implying multiple independent losses of the mitochondrial Tat pathway during the evolution of opisthokonts. In addition, bioinformatic evidence suggests that tatC comprises the entire Tat pathway in Oscarellidae, and that the Rieske Fe/S protein of mitochondrial complex III is its likely substrate.

Small inverted repeats drive mitochondrial genome evolution in Lake Baikal sponges.

Demosponges, the largest and most diverse class in the phylum Porifera, possess mitochondrial DNA (mtDNA) markedly different from that in other animals. Although several studies investigated evolution of demosponge mtDNA among major lineages of the group, the changes within these groups remain largely unexplored. Recently we determined mitochondrial genomic sequence of the Lake Baikal sponge Lubomirskia baicalensis and described proliferation of small inverted repeats (hairpins) that occurred in it since the divergence between L. baicalensis and the most closely related cosmopolitan freshwater sponge Ephydatia muelleri. Here we report mitochondrial genomes of three additional species of Lake Baikal sponges: Swartschewskia papyracea, Rezinkovia echinata and Baikalospongia intermedia morpha profundalis (Demospongiae, Haplosclerida, Lubomirskiidae) and from a more distantly related freshwater sponge Corvomeyenia sp. (Demospongiae, Haplosclerida, Metaniidae). We use these additional sequences to explore mtDNA evolution in Baikalian sponges, paying particular attention to the variation in the rates of nucleotide substitutions and the distribution of hairpins, abundant in these genomes. We show that most of the changes in Lubomirskiidae mitochondrial genomes are due to insertion/deletion/duplication of these elements rather than single nucleotide substitutions. Thus inverted repeats can act as an important force in evolution of mitochondrial genome architecture and be a valuable marker for population- and species-level studies in this group. In addition, we infer (((Rezinkovia+Lubomirskia)+Swartschewskia)+Baikalospongia) phylogeny for the family Lubomirskiidae based on the analysis of mitochondrial coding sequences from freshwater sponges.

Extreme mitochondrial evolution in the ctenophore Mnemiopsis leidyi: Insight from mtDNA and the nuclear genome.

Recent advances in sequencing technology have led to a rapid accumulation of mitochondrial DNA (mtDNA) sequences, which now represent the wide spectrum of animal diversity. However, one animal phylum--Ctenophora--has, to date, remained completely unsampled. Ctenophores, a small group of marine animals, are of interest due to their unusual biology, controversial phylogenetic position, and devastating impact as invasive species. Using data from the Mnemiopsis leidyi genome sequencing project, we Polymerase Chain Reaction (PCR) amplified and analyzed its complete mitochondrial (mt-) genome. At just over 10 kb, the mt-genome of M. leidyi is the smallest animal mtDNA ever reported and is among the most derived. It has lost at least 25 genes, including atp6 and all tRNA genes. We show that atp6 has been relocated to the nuclear genome and has acquired introns and a mitochondrial targeting presequence, while tRNA genes have been genuinely lost, along with nuclear-encoded mt-aminoacyl tRNA synthetases. The mt-genome of M. leidyi also displays extremely high rates of sequence evolution, which likely led to the degeneration of both protein and rRNA genes. In particular, encoded rRNA molecules possess little similarity with their homologs in other organisms and have highly reduced secondary structures. At the same time, nuclear encoded mt-ribosomal proteins have undergone expansions, likely to compensate for the reductions in mt-rRNA. The unusual features identified in M. leidyi mtDNA make this organism an interesting system for the study of various aspects of mitochondrial biology, particularly protein and tRNA import and mt-ribosome structures, and add to its value as an emerging model species. Furthermore, the fast-evolving M. leidyi mtDNA should be a convenient molecular marker for species- and population-level studies.