Convergent evolution of venom gland transcriptomes across Metazoa.

Animals have repeatedly evolved specialized organs and anatomical structures to produce and deliver a mixture of potent bioactive molecules to subdue prey or predators-venom. This makes it one of the most widespread, convergent functions in the animal kingdom. Whether animals have adopted the same genetic toolkit to evolved venom systems is a fascinating question that still eludes us. Here, we performed a comparative analysis of venom gland transcriptomes from 20 venomous species spanning the main Metazoan lineages to test whether different animals have independently adopted similar molecular mechanisms to perform the same function. We found a strong convergence in gene expression profiles, with venom glands being more similar to each other than to any other tissue from the same species, and their differences closely mirroring the species phylogeny. Although venom glands secrete some of the fastest evolving molecules (toxins), their gene expression does not evolve faster than evolutionarily older tissues. We found 15 venom gland-specific gene modules enriched in endoplasmic reticulum stress and unfolded protein response pathways, indicating that animals have independently adopted stress response mechanisms to cope with mass production of toxins. This, in turn, activates regulatory networks for epithelial development, cell turnover, and maintenance, which seem composed of both convergent and lineage-specific factors, possibly reflecting the different developmental origins of venom glands. This study represents a first step toward an understanding of the molecular mechanisms underlying the repeated evolution of one of the most successful adaptive traits in the animal kingdom.

The hourglass model of evolutionary conservation during embryogenesis extends to developmental enhancers with signatures of positive selection.

Inter-species comparisons of both morphology and gene expression within a phylum have revealed a period in the middle of embryogenesis with more similarity between species compared with earlier and later time points. This "developmental hourglass" pattern has been observed in many phyla, yet the evolutionary constraints on gene expression, as well as the underlying mechanisms of how this is regulated, remain elusive. Moreover, the role of positive selection on gene regulation in the more diverged earlier and later stages of embryogenesis remains unknown. Here, using DNase-seq to identify regulatory regions in two distant Drosophila species (D. melanogaster and D. virilis), we assessed the evolutionary conservation and adaptive evolution of enhancers throughout multiple stages of embryogenesis. This revealed a higher proportion of conserved enhancers at the phylotypic period, providing a regulatory basis for the hourglass expression pattern. Using an in silico mutagenesis approach, we detect signatures of positive selection on developmental enhancers at early and late stages of embryogenesis, with a depletion at the phylotypic period, suggesting positive selection as one evolutionary mechanism underlying the hourglass pattern of animal evolution.

Haplotype divergence supports long-term asexuality in the oribatid mite Oppiella nova.

Sex strongly impacts genome evolution via recombination and segregation. In the absence of these processes, haplotypes within lineages of diploid organisms are predicted to accumulate mutations independently of each other and diverge over time. This so-called "Meselson effect" is regarded as a strong indicator of the long-term evolution under obligate asexuality. Here, we present genomic and transcriptomic data of three populations of the asexual oribatid mite species Oppiella nova and its sexual relative Oppiella subpectinata We document strikingly different patterns of haplotype divergence between the two species, strongly supporting Meselson effect-like evolution and long-term asexuality in O. nova: I) variation within individuals exceeds variation between populations in O. nova but vice versa in O. subpectinata; II) two O. nova sublineages feature a high proportion of lineage-specific heterozygous single-nucleotide polymorphisms (SNPs), indicating that haplotypes continued to diverge after lineage separation; III) the deepest split in gene trees generally separates the two haplotypes in O. nova, but populations in O. subpectinata; and IV) the topologies of the two haplotype trees match each other. Our findings provide positive evidence for the absence of canonical sex over evolutionary time in O. nova and suggest that asexual oribatid mites can escape the dead-end fate usually associated with asexual lineages.

Rhythmicity is linked to expression cost at the protein level but to expression precision at the mRNA level.

Many genes have nycthemeral rhythms of expression, i.e. a 24-hours periodic variation, at either mRNA or protein level or both, and most rhythmic genes are tissue-specific. Here, we investigate and discuss the evolutionary origins of rhythms in gene expression. Our results suggest that rhythmicity of protein expression could have been favored by selection to minimize costs. Trends are consistent in bacteria, plants and animals, and are also supported by tissue-specific patterns in mouse. Unlike for protein level, cost cannot explain rhythm at the RNA level. We suggest that instead it allows to periodically reduce expression noise. Noise control had the strongest support in mouse, with limited evidence in other species. We have also found that genes under stronger purifying selection are rhythmically expressed at the mRNA level, and we propose that this is because they are noise sensitive genes. Finally, the adaptive role of rhythmic expression is supported by rhythmic genes being highly expressed yet tissue-specific. This provides a good evolutionary explanation for the observation that nycthemeral rhythms are often tissue-specific.

The Bgee suite: integrated curated expression atlas and comparative transcriptomics in animals.

Bgee is a database to retrieve and compare gene expression patterns in multiple animal species, produced by integrating multiple data types (RNA-Seq, Affymetrix, in situ hybridization, and EST data). It is based exclusively on curated healthy wild-type expression data (e.g., no gene knock-out, no treatment, no disease), to provide a comparable reference of normal gene expression. Curation includes very large datasets such as GTEx (re-annotation of samples as 'healthy' or not) as well as many small ones. Data are integrated and made comparable between species thanks to consistent data annotation and processing, and to calls of presence/absence of expression, along with expression scores. As a result, Bgee is capable of detecting the conditions of expression of any single gene, accommodating any data type and species. Bgee provides several tools for analyses, allowing, e.g., automated comparisons of gene expression patterns within and between species, retrieval of the prefered conditions of expression of any gene, or enrichment analyses of conditions with expression of sets of genes. Bgee release 14.1 includes 29 animal species, and is available at https://bgee.org/ and through its Bioconductor R package BgeeDB.

Special Care Is Needed in Applying Phylogenetic Comparative Methods to Gene Trees with Speciation and Duplication Nodes.

How gene function evolves is a central question of evolutionary biology. It can be investigated by comparing functional genomics results between species and between genes. Most comparative studies of functional genomics have used pairwise comparisons. Yet it has been shown that this can provide biased results, as genes, like species, are phylogenetically related. Phylogenetic comparative methods should be used to correct for this, but they depend on strong assumptions, including unbiased tree estimates relative to the hypothesis being tested. Such methods have recently been used to test the "ortholog conjecture," the hypothesis that functional evolution is faster in paralogs than in orthologs. Although pairwise comparisons of tissue specificity (τ) provided support for the ortholog conjecture, phylogenetic independent contrasts did not. Our reanalysis on the same gene trees identified problems with the time calibration of duplication nodes. We find that the gene trees used suffer from important biases, due to the inclusion of trees with no duplication nodes, to the relative age of speciations and duplications, to systematic differences in branch lengths, and to non-Brownian motion of tissue specificity on many trees. We find that incorrect implementation of phylogenetic method in empirical gene trees with duplications can be problematic. Controlling for biases allows successful use of phylogenetic methods to study the evolution of gene function and provides some support for the ortholog conjecture using three different phylogenetic approaches.

Identifying gene function and module connections by the integration of multispecies expression compendia.

The functions of many eukaryotic genes are still poorly understood. Here, we developed and validated a new method, termed GeneBridge, which is based on two linked approaches to impute gene function and bridge genes with biological processes. First, Gene-Module Association Determination (G-MAD) allows the annotation of gene function. Second, Module-Module Association Determination (M-MAD) allows predicting connectivity among modules. We applied the GeneBridge tools to large-scale multispecies expression compendia-1700 data sets with over 300,000 samples from human, mouse, rat, fly, worm, and yeast-collected in this study. G-MAD identifies novel functions of genes-for example, DDT in mitochondrial respiration and WDFY4 in T cell activation-and also suggests novel components for modules, such as for cholesterol biosynthesis. By applying G-MAD on data sets from respective tissues, tissue-specific functions of genes were identified-for instance, the roles of EHHADH in liver and kidney, as well as SLC6A1 in brain and liver. Using M-MAD, we identified a list of module-module associations, such as those between mitochondria and proteasome, mitochondria and histone demethylation, as well as ribosomes and lipid biosynthesis. The GeneBridge tools together with the expression compendia are available as an open resource, which will facilitate the identification of connections linking genes, modules, phenotypes, and diseases.

OMAmer: tree-driven and alignment-free protein assignment to subfamilies outperforms closest sequence approaches.

MOTIVATION: Assigning new sequences to known protein families and subfamilies is a prerequisite for many functional, comparative and evolutionary genomics analyses. Such assignment is commonly achieved by looking for the closest sequence in a reference database, using a method such as BLAST. However, ignoring the gene phylogeny can be misleading because a query sequence does not necessarily belong to the same subfamily as its closest sequence. For example, a hemoglobin which branched out prior to the hemoglobin alpha/beta duplication could be closest to a hemoglobin alpha or beta sequence, whereas it is neither. To overcome this problem, phylogeny-driven tools have emerged but rely on gene trees, whose inference is computationally expensive. RESULTS: Here, we first show that in multiple animal and plant datasets, 18-62% of assignments by closest sequence are misassigned, typically to an over-specific subfamily. Then, we introduce OMAmer, a novel alignment-free protein subfamily assignment method, which limits over-specific subfamily assignments and is suited to phylogenomic databases with thousands of genomes. OMAmer is based on an innovative method using evolutionarily informed k-mers for alignment-free mapping to ancestral protein subfamilies. Whilst able to reject non-homologous family-level assignments, we show that OMAmer provides better and quicker subfamily-level assignments than approaches relying on the closest sequence, whether inferred exactly by Smith-Waterman or by the fast heuristic DIAMOND. AVAILABILITYAND IMPLEMENTATION: OMAmer is available from the Python Package Index (as omamer), with the source code and a precomputed database available at https://github.com/DessimozLab/omamer. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

X chromosomes show relaxed selection and complete somatic dosage compensation across Timema stick insect species.

Sex chromosomes have evolved repeatedly across the tree of life. As they are present in different copy numbers in males and females, they are expected to experience different selection pressures than the autosomes, with consequences including a faster rate of evolution, increased accumulation of sexually antagonistic alleles and the evolution of dosage compensation. Whether these consequences are general or linked to idiosyncrasies of specific taxa is not clear as relatively few taxa have been studied thus far. Here, we use whole-genome sequencing to identify and characterize the evolution of the X chromosome in five species of Timema stick insects with XX:X0 sex determination. The X chromosome had a similar size (approximately 12% of the genome) and gene content across all five species, suggesting that the X chromosome originated prior to the diversification of the genus. Genes on the X showed evidence of relaxed selection (elevated dN/dS) and a slower evolutionary rate (dN + dS) than genes on the autosomes, likely due to sex-biased mutation rates. Genes on the X also showed almost complete dosage compensation in somatic tissues (heads and legs), but dosage compensation was absent in the reproductive tracts. Contrary to prediction, sex-biased genes showed little enrichment on the X, suggesting that the advantage X-linkage provides to the accumulation of sexually antagonistic alleles is weak. Overall, we found the consequences of X-linkage on gene sequences and expression to be similar across Timema species, showing the characteristics of the X chromosome are surprisingly consistent over 30 million years of evolution.

Dynamics of sex-biased gene expression during development in the stick insect Timema californicum.

Sexually dimorphic phenotypes are thought to arise primarily from sex-biased gene expression during development. Major changes in developmental strategies, such as the shift from hemimetabolous to holometabolous development, are therefore expected to have profound consequences for the dynamics of sex-biased gene expression. However, no studies have previously examined sex-biased gene expression during development in hemimetabolous insects, precluding comparisons between developmental strategies. Here we characterized sex-biased gene expression at three developmental stages in a hemimetabolous stick insect (Timema californicum): hatchlings, juveniles, and adults. As expected, the proportion of sex-biased genes gradually increased during development, mirroring the gradual increase of phenotypic sexual dimorphism. Sex-biased genes identified at early developmental stages were generally consistently male- or female-biased at later stages, suggesting their importance in sexual differentiation. Additionally, we compared the dynamics of sex-biased gene expression during development in T. californicum to those of the holometabolous fly Drosophila melanogaster by reanalyzing publicly available RNA-seq data from third instar larval, pupal and adult stages. In D. melanogaster, 84% of genes were sex-biased at the adult stage (compared to only 20% in T. californicum), and sex-biased gene expression increased abruptly at the adult stage when morphological sexual dimorphism is manifested. Our findings are consistent with the prediction that the dynamics of sex-biased gene expression during development differ extensively between holometabolous and hemimetabolous insect species.

Bio-SODA UX: enabling natural language question answering over knowledge graphs with user disambiguation.

The problem of natural language processing over structured data has become a growing research field, both within the relational database and the Semantic Web community, with significant efforts involved in question answering over knowledge graphs (KGQA). However, many of these approaches are either specifically targeted at open-domain question answering using DBpedia, or require large training datasets to translate a natural language question to SPARQL in order to query the knowledge graph. Hence, these approaches often cannot be applied directly to complex scientific datasets where no prior training data is available. In this paper, we focus on the challenges of natural language processing over knowledge graphs of scientific datasets. In particular, we introduce Bio-SODA, a natural language processing engine that does not require training data in the form of question-answer pairs for generating SPARQL queries. Bio-SODA uses a generic graph-based approach for translating user questions to a ranked list of SPARQL candidate queries. Furthermore, Bio-SODA uses a novel ranking algorithm that includes node centrality as a measure of relevance for selecting the best SPARQL candidate query. Our experiments with real-world datasets across several scientific domains, including the official bioinformatics Question Answering over Linked Data (QALD) challenge, as well as the CORDIS dataset of European projects, show that Bio-SODA outperforms publicly available KGQA systems by an F1-score of least 20% and by an even higher factor on more complex bioinformatics datasets. Finally, we introduce Bio-SODA UX, a graphical user interface designed to assist users in the exploration of large knowledge graphs and in dynamically disambiguating natural language questions that target the data available in these graphs.

Methods detecting rhythmic gene expression are biologically relevant only for strong signal.

The nycthemeral transcriptome embodies all genes displaying a rhythmic variation of their mRNAs periodically every 24 hours, including but not restricted to circadian genes. In this study, we show that the nycthemeral rhythmicity at the gene expression level is biologically functional and that this functionality is more conserved between orthologous genes than between random genes. We used this conservation of the rhythmic expression to assess the ability of seven methods (ARSER, Lomb Scargle, RAIN, JTK, empirical-JTK, GeneCycle, and meta2d) to detect rhythmic signal in gene expression. We have contrasted them to a naive method, not based on rhythmic parameters. By taking into account the tissue-specificity of rhythmic gene expression and different species comparisons, we show that no method is strongly favored. The results show that these methods designed for rhythm detection, in addition to having quite similar performances, are consistent only among genes with a strong rhythm signal. Rhythmic genes defined with a standard p-value threshold of 0.01 for instance, could include genes whose rhythmicity is biologically irrelevant. Although these results were dependent on the datasets used and the evolutionary distance between the species compared, we call for caution about the results of studies reporting or using large sets of rhythmic genes. Furthermore, given the analysis of the behaviors of the methods on real and randomized data, we recommend using primarily ARS, empJTK, or GeneCycle, which verify expectations of a classical distribution of p-values. Experimental design should also take into account the circumstances under which the methods seem more efficient, such as giving priority to biological replicates over the number of time-points, or to the number of time-points over the quality of the technique (microarray vs RNAseq). GeneCycle, and to a lesser extent empirical-JTK, might be the most robust method when applied to weakly informative datasets. Finally, our analyzes suggest that rhythmic genes are mainly highly expressed genes.

Genomic Features of Parthenogenetic Animals.

Evolution without sex is predicted to impact genomes in numerous ways. Case studies of individual parthenogenetic animals have reported peculiar genomic features that were suggested to be caused by their mode of reproduction, including high heterozygosity, a high abundance of horizontally acquired genes, a low transposable element load, or the presence of palindromes. We systematically characterized these genomic features in published genomes of 26 parthenogenetic animals representing at least 18 independent transitions to asexuality. Surprisingly, not a single feature was systematically replicated across a majority of these transitions, suggesting that previously reported patterns were lineage-specific rather than illustrating the general consequences of parthenogenesis. We found that only parthenogens of hybrid origin were characterized by high heterozygosity levels. Parthenogens that were not of hybrid origin appeared to be largely homozygous, independent of the cellular mechanism underlying parthenogenesis. Overall, despite the importance of recombination rate variation for the evolution of sexual animal genomes, the genome-wide absence of recombination does not appear to have had the dramatic effects which are expected from classical theoretical models. The reasons for this are probably a combination of lineage-specific patterns, the impact of the origin of parthenogenesis, and a survivorship bias of parthenogenetic lineages.

Inter-embryo gene expression variability recapitulates the hourglass pattern of evo-devo.

BACKGROUND: The evolution of embryological development has long been characterized by deep conservation. In animal development, the phylotypic stage in mid-embryogenesis is more conserved than either early or late stages among species within the same phylum. Hypotheses to explain this hourglass pattern have focused on purifying the selection of gene regulation. Here, we propose an alternative-genes are regulated in different ways at different stages and have different intrinsic capacities to respond to perturbations on gene expression. RESULTS: To eliminate the influence of natural selection, we quantified the expression variability of isogenetic single embryo transcriptomes throughout fly Drosophila melanogaster embryogenesis. We found that the expression variability is lower at the phylotypic stage, supporting that the underlying regulatory architecture in this stage is more robust to stochastic variation on gene expression. We present evidence that the phylotypic stage is also robust to genetic variations on gene expression. Moreover, chromatin regulation appears to play a key role in the variation and evolution of gene expression. CONCLUSIONS: We suggest that a phylum-level pattern of embryonic conservation can be explained by the intrinsic difference of gene regulatory mechanisms in different stages.

Correction to: Sex-specific changes in gene expression in response to estrogen pollution around the onset of sex differentiation in grayling (Salmonidae).

An amendment to this paper has been published and can be accessed via the original article.

Large-Scale Comparative Analysis of Codon Models Accounting for Protein and Nucleotide Selection.

There are numerous sources of variation in the rate of synonymous substitutions inside genes, such as direct selection on the nucleotide sequence, or mutation rate variation. Yet scans for positive selection rely on codon models which incorporate an assumption of effectively neutral synonymous substitution rate, constant between sites of each gene. Here we perform a large-scale comparison of approaches which incorporate codon substitution rate variation and propose our own simple yet effective modification of existing models. We find strong effects of substitution rate variation on positive selection inference. More than 70% of the genes detected by the classical branch-site model are presumably false positives caused by the incorrect assumption of uniform synonymous substitution rate. We propose a new model which is strongly favored by the data while remaining computationally tractable. With the new model we can capture signatures of nucleotide level selection acting on translation initiation and on splicing sites within the coding region. Finally, we show that rate variation is highest in the highly recombining regions, and we propose that recombination and mutation rate variation, such as high CpG mutation rate, are the two main sources of nucleotide rate variation. Although we detect fewer genes under positive selection in Drosophila than without rate variation, the genes which we detect contain a stronger signal of adaptation of dynein, which could be associated with Wolbachia infection. We provide software to perform positive selection analysis using the new model.

Repeated Evolution of Asexuality Involves Convergent Gene Expression Changes.

Asexual reproduction has evolved repeatedly from sexual ancestors across a wide range of taxa. Whereas the costs and benefits associated with asexuality have received considerable attention, the molecular changes underpinning the evolution of asexual reproduction remain relatively unexplored. In particular, it is completely unknown whether the repeated evolution of asexual phenotypes involves similar molecular changes, as previous studies have focused on changes occurring in single lineages. Here, we investigate the extent of convergent gene expression changes across five independent transitions to asexuality in stick insects. We compared gene expression of asexual females to females of close sexual relatives in whole-bodies, reproductive tracts, and legs. We identified a striking amount of convergent gene expression change (up to 8% of genes), greatly exceeding that expected by chance. Convergent changes were also tissue-specific, and most likely driven by selection for functional changes. Genes showing convergent changes in the reproductive tract were associated with meiotic spindle formation and centrosome organization. These genes are particularly interesting as they can influence the production of unreduced eggs, a key barrier to asexual reproduction. Changes in legs and whole-bodies were likely involved in female sexual trait decay, with enrichment in terms such as sperm-storage and pigmentation. By identifying changes occurring across multiple independent transitions to asexuality, our results provide a rare insight into the molecular basis of asexual phenotypes and suggest that the evolutionary path to asexuality is highly constrained, requiring repeated changes to the same key genes.

Cross-species functional modules link proteostasis to human normal aging.

The evolutionarily conserved nature of the few well-known anti-aging interventions that affect lifespan, such as caloric restriction, suggests that aging-related research in model organisms is directly relevant to human aging. Since human lifespan is a complex trait, a systems-level approach will contribute to a more comprehensive understanding of the underlying aging landscape. Here, we integrate evolutionary and functional information of normal aging across human and model organisms at three levels: gene-level, process-level, and network-level. We identify evolutionarily conserved modules of normal aging across diverse taxa, and notably show proteostasis to be conserved in normal aging. Additionally, we find that mechanisms related to protein quality control network are enriched for genes harboring genetic variants associated with 22 age-related human traits and associated to caloric restriction. These results demonstrate that a systems-level approach, combined with evolutionary conservation, allows the detection of candidate aging genes and pathways relevant to human normal aging.

Sex-specific changes in gene expression in response to estrogen pollution around the onset of sex differentiation in grayling (Salmonidae).

The synthetic 17α-ethinylestradiol (EE2) is a common estrogenic pollutant that has been suspected to affect the demography of river-dwelling salmonids. One possibility is that exposure to EE2 tips the balance during initial steps of sex differentiation, so that male genotypes show female-specific gene expression and gonad formation. Here we study EE2 effects on gene expression around the onset of sex differentiation in a population of European grayling (Thymallus thymallus) that suffers from sex ratio distortions. We exposed singly-raised embryos to one dose of 1 ng/L EE2, studied gene expression 10 days before hatching, at the day of hatching, and around the end of the yolk-sac stage, and related it to genetic sex (sdY genotype). We found that exposure to EE2 affects expression of a large number of genes, especially around hatching. These effects were strongly sex-dependent. We then raised fish for several months after hatching and found no evidence of sex reversal in the EE2-exposed fish. We conclude that ecologically relevant (i.e. low) levels of EE2 pollution do not cause sex reversal by simply tipping the balance at early stages of sex differentiation, but that they interfere with sex-specific gene expression.

Adaptive Evolution of Animal Proteins over Development: Support for the Darwin Selection Opportunity Hypothesis of Evo-Devo.

A driving hypothesis of evolutionary developmental biology is that animal morphological diversity is shaped both by adaptation and by developmental constraints. Here, we have tested Darwin's "selection opportunity" hypothesis, according to which high evolutionary divergence in late development is due to strong positive selection. We contrasted it to a "developmental constraint" hypothesis, according to which late development is under relaxed negative selection. Indeed, the highest divergence between species, both at the morphological and molecular levels, is observed late in embryogenesis and postembryonically. To distinguish between adaptation and relaxation hypotheses, we investigated the evidence of positive selection on protein-coding genes in relation to their expression over development, in fly Drosophila melanogaster, zebrafish Danio rerio, and mouse Mus musculus. First, we found that genes specifically expressed in late development have stronger signals of positive selection. Second, over the full transcriptome, genes with evidence for positive selection trend to be expressed in late development. Finally, genes involved in pathways with cumulative evidence of positive selection have higher expression in late development. Overall, there is a consistent signal that positive selection mainly affects genes and pathways expressed in late embryonic development and in adult. Our results imply that the evolution of embryogenesis is mostly conservative, with most adaptive evolution affecting some stages of postembryonic gene expression, and thus postembryonic phenotypes. This is consistent with the diversity of environmental challenges to which juveniles and adults are exposed.