Pseudofinder: Detection of Pseudogenes in Prokaryotic Genomes.

Prokaryotic genomes are usually densely packed with intact and functional genes. However, in certain contexts, such as after recent ecological shifts or extreme population bottlenecks, broken and nonfunctional gene fragments can quickly accumulate and form a substantial fraction of the genome. Identification of these broken genes, called pseudogenes, is a critical step for understanding the evolutionary forces acting upon, and the functional potential encoded within, prokaryotic genomes. Here, we present Pseudofinder, an open-source software dedicated to pseudogene identification and analysis in bacterial and archaeal genomes. We demonstrate that Pseudofinder's multi-pronged, reference-based approach can detect a wide variety of pseudogenes, including those that are highly degraded and typically missed by gene-calling pipelines, as well newly formed pseudogenes containing only one or a few inactivating mutations. Additionally, Pseudofinder can detect genes that lack inactivating substitutions but experiencing relaxed selection. Implementation of Pseudofinder in annotation pipelines will allow more precise estimations of the functional potential of sequenced microbes, while also generating new hypotheses related to the evolutionary dynamics of bacterial and archaeal genomes.

All essential endosymbionts of the ciliate Euplotes are cyclically replaced.

Symbiotic systems vary in the degree to which the partners are bound to each other1. At one extreme, there are intracellular endosymbionts in mutually obligate relationships with their host, often interpreted as mutualistic. The symbiosis between the betaproteobacterium Polynucleobacter and the ciliate Euplotes (clade B) challenges this view2: although freshwater Euplotes species long ago became dependent on endosymbionts, the many extant Polynucleobacter lineages they harbour arose recently and in parallel from different free-living ancestors2. The host requires the endosymbionts for reproduction and survival3, but each newly established symbiont is ultimately driven to extinction in a cycle of establishment, degeneration, and replacement. Similar replacement events have been observed in sap-feeding insects4-6, a model for bacteria-eukaryote symbioses7, but usually only affect a small subset of the host populations. Most insects retain an ancient coevolving symbiont, suggesting that long-term mutualism and permanent integration remain the rule and symbiont turnovers are mere evolutionary side-stories. Here we show that this is not the case for Euplotes. We examined all known essential Euplotes symbionts and found that none are ancient or coevolving; rather, all are recently established and continuously replaced over relatively short evolutionary time spans, making the symbiosis ancient for the host but not for any bacterial lineage.

What Can Environmental Sequences Tell Us About the Distribution of Low-Rank Taxa? The Case of Euplotes (Ciliophora, Spirotrichea), Including a Description of Euplotes enigma sp. nov.

Environmental sequences have become a major source of information. High-throughput sequencing (HTS) surveys have been used to infer biogeographic patterns and distribution of broad taxa of protists. This approach is, however, more questionable for addressing low-rank (less inclusive) taxa such as species and genera, because of the increased chance of errors in identification due to blurry taxonomic boundaries, low sequence divergence, or sequencing errors. The specious ciliate genus Euplotes partially escapes these limitations. It is a ubiquitous, monophyletic taxon, clearly differentiated from related genera, and with a relatively well-developed internal systematics. It has also been the focus of several ecological studies. We present an update on Euplotes biogeography, taking into consideration for the first time environmental sequences, both traditional (Sanger) and HTS. We inferred a comprehensive small subunit rRNA gene phylogeny of the genus including a newly described marine species, Euplotes enigma, characterized by a unique question mark-shaped macronucleus. We then added available environmental sequences to the tree, mapping associated metadata. The resulting scenario conflicts on many accounts with previously held views, suggesting, for example, that a large diversity of anaerobic Euplotes species exist, and that marine representatives of mainly freshwater lineages (and vice-versa) might be more common than previously thought.

EukRef-Ciliophora: a manually curated, phylogeny-based database of small subunit rRNA gene sequences of ciliates.

High-throughput sequencing (HTS) surveys, among the most common approaches currently used in environmental microbiology, require reliable reference databases to be correctly interpreted. The EukRef Initiative (eukref.org) is a community effort to manually screen available small subunit (SSU) rRNA gene sequences and produce a public, high-quality and informative framework of phylogeny-based taxonomic annotations. In the context of EukRef, we present a database for the monophyletic phylum Ciliophora, one of the most complex, diverse and ubiquitous protist groups. We retrieved more than 11 500 sequences of ciliates present in GenBank (28% from identified isolates and 72% from environmental surveys). Our approach included the inference of phylogenetic trees for every ciliate lineage and produced the largest SSU rRNA tree of the phylum Ciliophora to date. We flagged approximately 750 chimeric or low-quality sequences, improved the classification of 70% of GenBank entries and enriched environmental and literature metadata by 30%. The performance of EukRef-Ciliophora is superior to the current SILVA database in classifying HTS reads from a global marine survey. Comprehensive outputs are publicly available to make the new tool a useful guide for non-specialists and a quick reference for experts.

Biogeography and Character Evolution of the Ciliate Genus Euplotes (Spirotrichea, Euplotia), with Description of Euplotes curdsi sp. nov.

Ciliates comprise a diverse and ecologically important phylum of unicellular protists. One of the most specious and best-defined genera is Euplotes, which constitutes more than 70 morphospecies, many of which have never been molecularly tested. The increasing number of described Euplotes taxa emphasizes the importance for detailed characterizations of new ones, requiring standardized morphological observations, sequencing of molecular markers and careful comparison with previous literature. Here we describe Euplotes curdsi sp. nov., distinguishable by the combination of the following features: 45-65 µm length, oval or elongated shape with both ends rounded, narrow peristome with 25-34 adoral membranelles, conspicuous paroral membrane, double-eurystomus dorsal argyrome type, 6-7 dorsolateral kineties and 10 frontoventral cirri. Three populations of the novel species have been found in brackish and marine samples in the Mediterranean and the White Sea. We provide the SSU rRNA gene sequences of these populations, and an updated phylogeny of the genus Euplotes. Using the molecular phylogenetic tree, we inferred aspects of the biogeographical history of the genus and the evolution of its most important taxonomic characters in order to provide a frame for future descriptions. Ultimately, these data reveal recurrent trends of freshwater invasion and highlight the dynamic, yet convergent, morphological evolution of Euplotes.