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Reproductive isolation and genomic divergence both accumulate over time in the formation and persistence of distinct biological species. The pace of “speciation clocks” quantified with pre-zygotic and post-zygotic reproductive isolation, however, differs among taxa, with pre-zygotic isolation tending to evolve sooner in some but not all taxa. To address this issue in nematodes for the first time, here we infer the species tree and divergence times across the phylogeny of 51 species of Caenorhabditis . We incorporate several molecular evolutionary strategies in phylogenomic dating to account for complications in this group due to lack of fossil calibration, deep molecular divergence with synonymous-site saturation, and codon usage bias. By integrating divergence times with experimental data on pre- and post-zygotic reproductive isolation, we infer that post-zygotic isolation accumulates faster than pre-zygotic isolation in Caenorhabditis and that hybrid sterility evolves sooner than hybrid inviability. These findings are consistent with speciation being driven principally by intrinsic isolating barriers and the disproportionate fragility of germline developmental programs to disruption. We estimate that it takes approximately 50 million generations for intrinsic post-zygotic reproductive compatibility to be reduced by half, on average, between diverging pairs of Caenorhabditis . The protracted reproductive isolation clocks in Caenorhabditis may, in part, reflect the capacity to retain population genetic hyperdiversity, the incomplete sampling of global biodiversity, and as-yet uncharacterized incipient or cryptic species.

[This corrects the article DOI: 10.1371/journal.pcbi.1012208.].

The apicomplexan intracellular parasite Toxoplasma gondii is a major food borne pathogen that is highly prevalent in the global population. The majority of the T . gondii proteome remains uncharacterized and the organization of proteins into complexes is unclear. To overcome this knowledge gap, we used a biochemical fractionation strategy to predict interactions by correlation profiling. To overcome the deficit of high-quality training data in non-model organisms, we complemented a supervised machine learning strategy, with an unsupervised approach, based on similarity network fusion. The resulting combined high confidence network, ToxoNet, comprises 2,063 interactions connecting 652 proteins. Clustering identifies 93 protein complexes. We identified clusters enriched in mitochondrial machinery that include previously uncharacterized proteins that likely represent novel adaptations to oxidative phosphorylation. Furthermore, complexes enriched in proteins localized to secretory organelles and the inner membrane complex, predict additional novel components representing novel targets for detailed functional characterization. We present ToxoNet as a publicly available resource with the expectation that it will help drive future hypotheses within the research community.

Abstract Geographically distinct populations can adapt to the temperature conditions of their local environment, leading to temperature-dependent fitness differences between populations. Consistent with local adaptation, phylogeographically distinct Caenorhabditis briggsae nematodes show distinct fitness responses to temperature. The genetic mechanisms underlying local adaptation, however, remain unresolved. To investigate the potential role of small noncoding RNAs in genotype-specific responses to temperature, we quantified small RNA expression using high-throughput sequencing of C. briggsae nematodes from tropical and temperate strain genotypes reared under three temperature conditions (14 °C, 20 °C, and 30 C). Strains representing both tropical and temperate regions showed significantly lower expression of PIWI-interacting RNAs (piRNAs) at high temperatures, primarily mapping to a large ∼7 Mb long piRNA cluster on chromosome IV. We also documented decreased expression of 22G-RNAs antisense to protein-coding genes and other genomic features at high rearing temperatures for the thermally-intolerant temperate strain genotype, but not for the tropical strain genotype. Reduced 22G-RNA expression was widespread along chromosomes and among feature types, indicative of a genome-wide response. Targets of the EGO-1/CSR-1 22G-RNA pathway were most strongly impacted compared with other 22G-RNA pathways, implicating the CSR-1 Argonaute and its RNA-dependent RNA polymerase EGO-1 in the genotype-dependent modulation of C. briggsae 22G-RNAs under chronic thermal stress. Our work suggests that gene regulation via small RNAs may be an important contributor to the evolution of local adaptations.

Geographically distinct populations can adapt to the temperature conditions of their local environment, leading to temperature-dependent fitness differences between populations. Consistent with local adaptation, phylogeographically distinct Caenorhabditis briggsae nematodes show distinct fitness responses to temperature. The genetic mechanisms underlying local adaptation, however, remain unresolved. To investigate the potential role of small noncoding RNAs in genotype-specific responses to temperature, we quantified small RNA expression using high-throughput sequencing of C. briggsae nematodes from tropical and temperate strain genotypes reared under three temperature conditions (14˚C, 20˚C, 30˚C). Strains representing both tropical and temperate regions showed significantly lower expression of PIWI-interacting RNAs (piRNAs) at high temperatures, primarily mapping to a large ~7 Mb long piRNA cluster on chromosome IV. We also documented decreased expression of 22G-RNAs antisense to protein-coding genes and other genomic features at high rearing temperatures for the thermally-intolerant temperate strain genotype, but not for the tropical strain genotype. Reduced 22G-RNA expression was widespread along chromosomes and among feature types, indicative of a genome-wide response. Targets of the EGO-1/CSR-1 22G-RNA pathway were most strongly impacted compared to other 22G-RNA pathways, implicating the CSR-1 Argonaute and its RNA-dependent RNA polymerase EGO-1 in the genotype-dependent modulation of C. briggsae 22G-RNAs under chronic thermal stress. Our work suggests that gene regulation via small RNAs may be an important contributor to the evolution of local adaptations. Competing Interest Statement The authors have declared no competing interest.

Diverse regulatory mechanisms enable precise spatio-temporal control of gene expression across developmental stages, tissues, and sexes, contributing to the proper development of the organism. Evolutionary divergence leads to species-specific gene expression patterns, even in preserved developmental structures, due to regulatory changes that can disproportionately influence subsets of developmental genetic networks. Here we quantify the evolution of sex-biased and tissue-biased transcriptomes from two tissue types (gonad and soma) for each of two sexes (male and female) from two of the closest known sister species of Caenorhabditis nematodes (C. remanei and C. latens). Differential gene expression and co-expression network analyses identify gene sets with distinct transcriptomic profiles, revealing widespread divergence between these morphologically and developmentally cryptic sister species. The transcriptomic divergence occurs despite most genes showing conserved expression across tissues and sexes. These observations implicate shared selection pressures related to tissue and sex differences as outweighing species-specific selection and developmental system drift in shaping overall transcriptome profiles. Although developmentally-plastic tissue-biased expression profiles are mostly conserved between species, we find that sex-biased genes, particularly male-biased genes, contribute disproportionately to species-differences in gene expression, consistent with a disproportionate role for male-biased selection driving gene expression divergence.

Diverse small RNA pathways, comprised of Argonaute effector proteins and their bound small RNA molecules, define critical systems for regulating gene expression in all domains of life. Some small RNA pathways have undergone significant evolutionary change in nematode roundworms, including gains of novel Argonaute genes and losses of entire pathways. Differences in the functional complement of Argonautes among species therefore profoundly influence the available repertoire of mechanisms for gene regulation. Despite intensive study of Argonaute function in Caenorhabditis elegans, the extent of Argonaute gene family dynamism and functional breadth remains unknown. We therefore comprehensively surveyed Argonautes across 51 Caenorhabditis species, yielding over 1200 genes from 11 subfamilies. We documented multiple cases of diversification, including the birth of a potentially novel Argonaute subfamily and the origin of the ALG-5 microRNA Argonaute near the base of the Caenorhabditis phylogeny, as well as evidence of adaptive sequence evolution and gain of a new splice isoform for CSR-1 in a clade of 31 species. We also detected repeated independent losses of multiple components of the piRNA pathway, mirroring other instances of piRNA pathway loss across the phylum. Gene gain and loss occurs significantly faster than expected within several Argonaute subfamilies, potentially associated with transposable element proliferation coevolving with WAGO-9/10/12 copy number variation. Our characterization of Argonaute diversity across Caenorhabditis demonstrates exceptional functional dynamism in the evolution of gene regulation, with broad implications for mechanisms of control over ontogenetic development and genome integrity. For organisms to develop properly to survive and reproduce, they must express their genes in the right amount, in the appropriate cell types and time during development. One important mechanism that organisms use to regulate gene expression involves small RNA pathways, where short molecules of RNA serve as targeting guides by binding to Argonaute effector proteins. To understand how small RNA pathways evolve over time, we searched for Argonaute genes throughout the genomes of 51 species of Caenorhabditis nematode worms and found over 1200 Argonaute genes belonging to 11 different Argonaute subfamilies. We then documented cases where species have evolved potentially new types of Argonautes, or new protein isoforms of existing Argonautes. We also identified repeated cases of evolutionary loss of entire Argonaute subfamilies, including for the PRG-1 Argonaute needed in the piRNA regulatory pathway, and characterized how some Argonaute subfamilies gain and lose genes significantly faster than expected. Our findings demonstrate substantial variation in the functional repertoire of Argonaute genes found among Caenorhabditis species, with this evolutionary dynamism implicating fundamental differences between species in how they regulate gene expression across their genomes throughout development.

Introns play a critical role in regulating alternative splicing. However, identifying functional intronic motifs is challenging due to their short and degenerate sequence composition. Massively parallel reporter assays have provided insights into cis-regulatory logic governing alternative splicing, but these approaches are generally performed in cell culture, limiting their ability to capture tissue-specific contexts. Here, we implemented in vivo Parallelized Reporter Assays in C. elegans neurons and muscle cells to screen for intronic enhancer and silencer motifs among thousands of randomized sequences. We identified nearly 200 sequences regulating splicing in these tissues. We uncovered core sub-sequences with tissue-biased enhancing and silencing activity, including motifs recognized by well-characterized RNA-binding proteins, and orphan motifs with no obvious cognate binding protein. Mapping our PRA-derived motifs to native introns flanking tissue-biased alternative exons revealed their conservation across nematodes, supporting their functional relevance. Additionally, individual intronic regions frequently contained diverse combinations of these motifs, indicative of complex engagement of these sequences by RNA-binding proteins. Finally, we performed targeted mutagenesis of PRA-derived intronic enhancers flanking a neuronal microexon, identifying key cis-regulatory determinants of microexon splicing. Together, our study provides a framework to explore the role of intronic regions in tissue-specific splicing regulation within a multicellular organism.