CaeNDR, the Caenorhabditis Natural Diversity Resource.

Studies of model organisms have provided important insights into how natural genetic differences shape trait variation. These discoveries are driven by the growing availability of genomes and the expansive experimental toolkits afforded to researchers using these species. For example, Caenorhabditis elegans is increasingly being used to identify and measure the effects of natural genetic variants on traits using quantitative genetics. Since 2016, the C. elegans Natural Diversity Resource (CeNDR) has facilitated many of these studies by providing an archive of wild strains, genome-wide sequence and variant data for each strain, and a genome-wide association (GWA) mapping portal for the C. elegans community. Here, we present an updated platform, the Caenorhabditis Natural Diversity Resource (CaeNDR), that enables quantitative genetics and genomics studies across the three Caenorhabditis species: C. elegans, C. briggsae and C. tropicalis. The CaeNDR platform hosts several databases that are continually updated by the addition of new strains, whole-genome sequence data and annotated variants. Additionally, CaeNDR provides new interactive tools to explore natural variation and enable GWA mappings. All CaeNDR data and tools are accessible through a freely available web portal located at caendr.org.

Additional description and genome analyses of Caenorhabditis auriculariae representing the basal lineage of genus Caenorhabditis.

Caenorhabditis auriculariae, which was morphologically described in 1999, was re-isolated from a Platydema mushroom-associated beetle. Based on the re-isolated materials, some morphological characteristics were re-examined and ascribed to the species. In addition, to clarify phylogenetic relationships with other Caenorhabditis species and biological features of the nematode, the whole genome was sequenced and assembled into 109.5 Mb with 16,279 predicted protein-coding genes. Molecular phylogenetic analyses based on ribosomal RNA and 269 single-copy genes revealed the species is closely related to C. sonorae and C. monodelphis placing them at the most basal clade of the genus. C. auriculariae has morphological characteristics clearly differed from those two species and harbours a number of species-specific gene families, indicating its usefulness as a new outgroup species for Caenorhabditis evolutionary studies. A comparison of carbohydrate-active enzyme (CAZy) repertoires in genomes, which we found useful to speculate about the lifestyle of Caenorhabditis nematodes, suggested that C. auriculariae likely has a life-cycle with tight-association with insects.

Large genetic diversity and strong positive selection in F-box and GPCR genes among the wild isolates of Caenorhabditis elegans.

The F-box and chemosensory GPCR (csGPCR) gene families are greatly expanded in nematodes, including the model organism Caenorhabditis elegans, compared to insects and vertebrates. However, the intraspecific evolution of these two gene families in nematodes remain unexamined. In this study, we analyzed the genomic sequences of 330 recently sequenced wild isolates of C. elegans using a range of population genetics approaches. We found that F-box and csGPCR genes, especially the Srw family csGPCRs, showed much more diversity than other gene families. Population structure analysis and phylogenetic analysis divided the wild strains into eight non-Hawaiian and three Hawaiian subpopulations. Some Hawaiian strains appeared to be more ancestral than all other strains. F-box and csGPCR genes maintained a great amount of the ancestral variants in the Hawaiian subpopulation and their divergence among the non-Hawaiian subpopulations contributed significantly to population structure. F-box genes are mostly located at the chromosomal arms and high recombination rate correlates with their large polymorphism. Moreover, using both neutrality tests and Extended Haplotype Homozygosity analysis, we identified signatures of strong positive selection in the F-box and csGPCR genes among the wild isolates, especially in the non-Hawaiian population. Accumulation of high-frequency derived alleles in these genes was found in non-Hawaiian population, leading to divergence from the ancestral genotype. In summary, we found that F-box and csGPCR genes harbour a large pool of natural variants, which may be subjected to positive selection. These variants are mostly mapped to the substrate-recognition domains of F-box proteins and the extracellular and intracellular regions of csGPCRs, possibly resulting in advantages during adaptation by affecting protein degradation and the sensing of environmental cues, respectively.

Ubiquitous Selfish Toxin-Antidote Elements in Caenorhabditis Species.

Toxin-antidote elements (TAs) are selfish genetic dyads that spread in populations by selectively killing non-carriers. TAs are common in prokaryotes, but very few examples are known in animals. Here, we report the discovery of maternal-effect TAs in both C. tropicalis and C. briggsae, two distant relatives of C. elegans. In C. tropicalis, multiple TAs combine to cause a striking degree of intraspecific incompatibility: five elements reduce the fitness of >70% of the F2 hybrid progeny of two Caribbean isolates. We identified the genes underlying one of the novel TAs, slow-1/grow-1, and found that its toxin, slow-1, is homologous to nuclear hormone receptors. Remarkably, although previously known TAs act during embryonic development, maternal loading of slow-1 in oocytes specifically slows down larval development, delaying the onset of reproduction by several days. Finally, we found that balancing selection acting on linked, conflicting TAs hampers their ability to spread in populations, leading to more stable genetic incompatibilities. Our findings indicate that TAs are widespread in Caenorhabditis species and target a wide range of developmental processes and that antagonism between them may cause lasting incompatibilities in natural populations. We expect that similar phenomena exist in other animal species.

Chromosome-Level Assembly of the Caenorhabditis remanei Genome Reveals Conserved Patterns of Nematode Genome Organization.

The nematode Caenorhabditis elegans is one of the key model systems in biology, including possessing the first fully assembled animal genome. Whereas C. elegans is a self-reproducing hermaphrodite with fairly limited within-population variation, its relative C. remanei is an outcrossing species with much more extensive genetic variation, making it an ideal parallel model system for evolutionary genetic investigations. Here, we greatly improve on previous assemblies by generating a chromosome-level assembly of the entire C. remanei genome (124.8 Mb of total size) using long-read sequencing and chromatin conformation capture data. Like other fully assembled genomes in the genus, we find that the C. remanei genome displays a high degree of synteny with C. elegans despite multiple within-chromosome rearrangements. Both genomes have high gene density in central regions of chromosomes relative to chromosome ends and the opposite pattern for the accumulation of repetitive elements. C. elegans and C. remanei also show similar patterns of interchromosome interactions, with the central regions of chromosomes appearing to interact with one another more than the distal ends. The new C. remanei genome presented here greatly augments the use of the Caenorhabditis as a platform for comparative genomics and serves as a basis for molecular population genetics within this highly diverse species.

The Genome of Caenorhabditis bovis.

The free-living nematode Caenorhabditis elegans is a key laboratory model for metazoan biology. C. elegans has also become a model for parasitic nematodes despite being only distantly related to most parasitic species. All of the ∼65 Caenorhabditis species currently in culture are free-living, with most having been isolated from decaying plant or fungal matter. Caenorhabditis bovis is a particularly unusual species that has been isolated several times from the inflamed ears of Zebu cattle in Eastern Africa, where it is associated with the disease bovine parasitic otitis. C. bovis is therefore of particular interest to researchers interested in the evolution of nematode parasitism. However, as C. bovis is not in laboratory culture, it remains little studied. Here, by sampling livestock markets and slaughterhouses in Western Kenya, we successfully reisolated C. bovis from the ear of adult female Zebu. We sequenced the genome of C. bovis using the Oxford Nanopore MinION platform in a nearby field laboratory and used the data to generate a chromosome-scale draft genome sequence. We exploited this draft genome sequence to reconstruct the phylogenetic relationships of C. bovis to other Caenorhabditis species and reveal the changes in genome size and content that have occurred during its evolution. We also identified expansions in several gene families that have been implicated in parasitism in other nematode species. The high-quality draft genome and our analyses thereof represent a significant advancement in our understanding of this unusual Caenorhabditis species.

Origin and Evolution of Two Independently Duplicated Genes Encoding UDP- Glucose: Glycoprotein Glucosyltransferases in Caenorhabditis and Vertebrates.

UDP- glucose: glycoprotein glucosyltransferase (UGGT) is a protein that operates as the gatekeeper for the endoplasmic reticulum (ER) quality control mechanism of glycoprotein folding. It is known that vertebrates and Caenorhabditis genomes harbor two uggt gene copies that exhibit differences in their properties.Bayesian phylogenetic inference based on 195 UGGT and UGGT-like protein sequences of an ample spectrum of eukaryotic species showed that uggt genes went through independent duplications in Caenorhabditis and vertebrates. In both lineages, the catalytic domain of the duplicated genes was subjected to a strong purifying selective pressure, while the recognition domain was subjected to episodic positive diversifying selection. Selective relaxation in the recognition domain was more pronounced in Caenorhabditis uggt-b than in vertebrates uggt-2 Structural bioinformatics analysis revealed that Caenorhabditis UGGT-b protein lacks essential sequences proposed to be involved in the recognition of unfolded proteins. When we assayed glucosyltrasferase activity of a chimeric protein composed by Caenorhabditis uggt-b recognition domain fused to S. pombe catalytic domain expressed in yeast, no activity was detected.The present results support the conservation of the UGGT activity in the catalytic domain and a putative divergent function of the recognition domain for the UGGT2 protein in vertebrates, which would have gone through a specialization process. In Caenorhabditis, uggt-b evolved under different constraints compared to uggt-a which, by means of a putative neofunctionalization process, resulted in a non-redundant paralog. The non-canonical function of uggt-b in the worm lineage highlights the need to take precautions before generalizing gene functions in model organisms.

Artificial selection for increased dispersal results in lower fitness.

Dispersal often covaries with other traits, and this covariation was shown to have a genetic basis. Here, we wanted to explore to what extent genetic constraints and correlational selection can explain patterns of covariation between dispersal and key life-history traits-lifespan and reproduction. A prediction from the fitness-associated dispersal hypothesis was that lower genetic quality is associated with higher dispersal propensity as driven by the benefits of genetic mixing. We wanted to contrast it with a prediction from a different model that individuals putting more emphasis on current rather than future reproduction disperse more, as they are expected to be more risk-prone and exploratory. However, if dispersal has inherent costs, this will also result in a negative genetic correlation between higher rates of dispersal and some aspects of performance. To explore this issue, we used the dioecious nematode Caenorhabditis remanei and selected for increased and decreased dispersal propensity for 10 generations, followed by five generations of relaxed selection. Dispersal propensity responded to selection, and females from high-dispersal lines dispersed more than females from low-dispersal lines. Females selected for increased dispersal propensity produced fewer offspring and were more likely to die from matricide, which is associated with a low physiological condition in Caenorhabditis nematodes. There was no evidence for differences in age-specific reproductive effort between high- and low-dispersal females. Rather, reproductive output of high-dispersal females was consistently reduced. We argue that our data provide support for the fitness-associated dispersal hypothesis.

Noda-Like RNA Viruses Infecting Caenorhabditis Nematodes: Sympatry, Diversity, and Reassortment.

Three RNA viruses related to nodaviruses were previously described to naturally infect the nematode Caenorhabditis elegans and its relative, Caenorhabditis briggsae Here, we report on a collection of more than 50 viral variants from wild-caught Caenorhabditis. We describe the discovery of a new related virus, the Melník virus, infecting C. briggsae, which similarly infects intestinal cells. In France, a frequent pattern of coinfection of C. briggsae by the Santeuil virus and Le Blanc virus was observed at the level of an individual nematode and even a single cell. We do not find evidence of reassortment between the RNA1 and RNA2 molecules of Santeuil and Le Blanc viruses. However, by studying patterns of evolution of each virus, reassortments of RNA1 and RNA2 among variants of each virus were identified. We develop assays to test the relative infectivity and competitive ability of the viral variants and detect an interaction between host genotype and Santeuil virus genotype, such that the result depends on the host strain.IMPORTANCE The roundworm Caenorhabditis elegans is a laboratory model organism in biology. We study natural populations of this small animal and its relative, C. briggsae, and the viruses that infect them. We previously discovered three RNA viruses related to nodaviruses and here describe a fourth one, called the Melník virus. These viruses have a genome composed of two RNA molecules. We find that two viruses may infect the same animal and the same cell. The two RNA molecules may be exchanged between variants of a given viral species. We study the diversity of each viral species and devise an assay of their infectivity and competitive ability. Using this assay, we show that the outcome of the competition also depends on the host.

Telomeric small RNAs in the genus Caenorhabditis.

Telomeric DNA is composed of simple tandem repeat sequences and has a G-rich strand that runs 5' to 3' toward the chromosome terminus. Small RNAs with homology to telomeres have been observed in several organisms and could originate from telomeres or from interstitial telomere sequences (ITSs), which are composites of degenerate and perfect telomere repeat sequences found on chromosome arms. We identified Caenorhabditis elegans small RNAs composed of the Caenorhabditis telomere sequence (TTAGGC)n with up to three mismatches, which might interact with telomeres. We rigorously defined ITSs for genomes of C. elegans and for two closely related nematodes, Caenorhabditis briggsae and Caenorhabditis remanei Most telomeric small RNAs with mismatches originated from ITSs, which were depleted from mRNAs but were enriched in introns whose genes often displayed hallmarks of genomic silencing. C. elegans small RNAs composed of perfect telomere repeats were very rare but their levels increased by several orders of magnitude in C. briggsae and C. remanei Major small RNA species in C. elegans begin with a 5' guanine nucleotide, which was strongly depleted from perfect telomeric small RNAs of all three Caenorhabditis species. Perfect G-rich or C-rich telomeric small RNAs commonly began with 5' UAGGCU and 5' UUAGGC or 5' CUAAGC, respectively. In contrast, telomeric small RNAs with mismatches had a mixture of all four 5' nucleotides. We suggest that perfect telomeric small RNAs have a mechanism of biogenesis that is distinct from known classes of small RNAs and that a dramatic change in their regulation occurred during recent Caenorhabditis evolution.

Transcriptomic analyses reveal groups of co-expressed, syntenic lncRNAs in four species of the genus Caenorhabditis.

Long non-coding RNAs (lncRNAs) are a heterogeneous class of genes that do not code for proteins. Since lncRNAs (or a fraction thereof) are expected to be functional, many efforts have been dedicated to catalog lncRNAs in numerous organisms, but our knowledge of lncRNAs in non vertebrate species remains very limited. Here, we annotated lncRNAs using transcriptomic data from the same larval stage of four Caenorhabditis species. The number of annotated lncRNAs in self-fertile nematodes was lower than in out-crossing species. We used a combination of approaches to identify putatively homologous lncRNAs: synteny, sequence conservation, and structural conservation. We classified a total of 1,532 out of 7,635 genes from the four species into families of lncRNAs with conserved synteny and expression at the larval stage, suggesting that a large fraction of the predicted lncRNAs may be species specific. Despite both sequence and local secondary structure seem to be poorly conserved, sequences within families frequently shared BLASTn hits and short sequence motifs, which were more likely to be unpaired in the predicted structures. We provide the first multi-species catalog of lncRNAs in nematodes and identify groups of lncRNAs with conserved synteny and expression, that share exposed motifs.

Genetic basis to hybrid inviability is more complex than hybrid male sterility in Caenorhabditis nematodes.

Hybrid male sterility often evolves before female sterility or inviability of hybrids, implying that the accumulation of divergence between separated lineages should lead hybrid male sterility to have a more polygenic basis. However, experimental evidence is mixed. Here, we use the nematodes Caenorhabditis remanei and C. latens to characterize the underlying genetic basis of asymmetric hybrid male sterility and hybrid inviability. We demonstrate that hybrid male sterility is consistent with a simple genetic basis, involving a single X-autosome incompatibility. We also show that hybrid inviability involves more genomic compartments, involving diverse nuclear-nuclear incompatibilities, a mito-nuclear incompatibility, and maternal effects. These findings demonstrate that male sensitivity to genetic perturbation may be genetically simple compared to hybrid inviability in Caenorhabditis and motivates tests of generality for the genetic architecture of hybrid incompatibility across the breadth of phylogeny.

Genomic basis of recombination suppression in the hybrid between Caenorhabditis briggsae and C. nigoni.

DNA recombination is required for effective segregation and diversification of genomes and for the successful completion of meiosis. Recent studies in various species hybrids have demonstrated a genetic link between DNA recombination and speciation. Consistent with this, we observed a striking suppression of recombination in the hybrids between two nematodes, the hermaphroditic Caenorhabditis briggsae and the gonochoristic C. nigoni. To unravel the molecular basis underlying the recombination suppression in their hybrids, we generated a C. nigoni genome with chromosome-level contiguity and produced an improved C. briggsae genome with resolved gaps up to 2.8 Mb. The genome alignment reveals not only high sequence divergences but also pervasive intra- and inter-chromosomal sequence re-arrangements between the two species, which are plausible culprits for the observed suppression. Comparison of recombination boundary sequences suggests that recombination in the hybrid requires extensive sequence homology, which is rarely seen between the two genomes. The new genomes and genomic libraries form invaluable resources for studying genome evolution, hybrid incompatibilities and sex evolution for this pair of model species.

Rapid genome shrinkage in a self-fertile nematode reveals sperm competition proteins.

To reveal impacts of sexual mode on genome content, we compared chromosome-scale assemblies of the outcrossing nematode Caenorhabditis nigoni to its self-fertile sibling species, C. briggsaeC. nigoni's genome resembles that of outcrossing relatives but encodes 31% more protein-coding genes than C. briggsaeC. nigoni genes lacking C. briggsae orthologs were disproportionately small and male-biased in expression. These include the male secreted short (mss) gene family, which encodes sperm surface glycoproteins conserved only in outcrossing species. Sperm from mss-null males of outcrossing C. remanei failed to compete with wild-type sperm, despite normal fertility in noncompetitive mating. Restoring mss to C. briggsae males was sufficient to enhance sperm competitiveness. Thus, sex has a pervasive influence on genome content that can be used to identify sperm competition factors.

Differing roles for sur-2/MED23 in C. elegans and C. briggsae vulval development.

Normal vulval development in the nematode Caenorhabditis briggsae is identical to that in the related Caenorhabditis elegans. However, several experiments suggest that there are differences between the two species with respect to the contribution of EGF/Ras signaling. To investigate these differences genetically, we have characterized a C. briggsae mutant strain that phenocopies the effect observed when C. briggsae animals are treated with U0126, an inhibitor of the EGF pathway component MEK. We identify that the gene affected in the mutant strain is Cbr-sur-2, which encodes a MED23 mediator complex protein that acts downstream of EGF signaling in C. elegans and other organisms, such as mammals. When Cbr-sur-2 and Cel-sur-2 mutants are compared, we find that the production of additional vulval cells from P5.p and P7.p in C. elegans is dependent on proper development of P6.p, while C. briggsae does not have a similar requirement. Combined chemical and genetic interference with the EGF pathway completely eliminates vulval development in C. elegans but not in C. briggsae. Our results provide genetic evidence for the differing requirements for EGF signaling in the two species.

Impact of genetic background and experimental reproducibility on identifying chemical compounds with robust longevity effects.

Limiting the debilitating consequences of ageing is a major medical challenge of our time. Robust pharmacological interventions that promote healthy ageing across diverse genetic backgrounds may engage conserved longevity pathways. Here we report results from the Caenorhabditis Intervention Testing Program in assessing longevity variation across 22 Caenorhabditis strains spanning 3 species, using multiple replicates collected across three independent laboratories. Reproducibility between test sites is high, whereas individual trial reproducibility is relatively low. Of ten pro-longevity chemicals tested, six significantly extend lifespan in at least one strain. Three reported dietary restriction mimetics are mainly effective across C. elegans strains, indicating species and strain-specific responses. In contrast, the amyloid dye ThioflavinT is both potent and robust across the strains. Our results highlight promising pharmacological leads and demonstrate the importance of assessing lifespans of discrete cohorts across repeat studies to capture biological variation in the search for reproducible ageing interventions.

Conservation of anatomically restricted glycosaminoglycan structures in divergent nematode species.

Heparan sulfates (HS) are glycosaminoglycans of the extracellular matrices and characterized by complex modification patterns owing to sulfations, epimerization, and acetylation. Distinct HS modification patterns have been shown to modulate protein-protein interactions during development in general and of the nervous system in particular. This has led to the heparan sulfate code hypothesis, which posits that specifically modified HS epitopes are distributed in a tissue and cell-specific fashion to orchestrate neural circuit formation. Whether an HS code exists in vivo, how specific or how evolutionarily conserved the anatomical distribution of an HS code may be has remained unknown. Here we conduct a systematic comparison of HS modification patterns in the nematode Caenorhabditis elegans using transgenic expression of 33 different HS-specific single chain variable fragment antibodies. We find that some HS modification patterns are widely distributed in the nervous system. In contrast, other HS modification patterns appear highly cell-specific in both non-neuronal and neuronal cells. Some patterns can be as restricted in their localization as to single neurites or synaptic connections between two neurons. This restricted anatomical localization of specific HS patterns can be evolutionarily conserved over a span of 80-100 million years in the divergent nematode species Caenorhabditis briggsae suggesting structural and, possibly functional conservation of glycosaminoglycan structures similar to proteins. These findings suggest a HS code with subcellularly localized, unique glycan identities in the nervous system.

Mitochondrial genome of Caenorhabditis nigoni (Rhabditida: Rhabditidae).

To facilitate comparative genomic study in the Caenorhabditis species, the mitochondrial genome (mitogenome) of a nematode species Caenorhabditis nigoni (previous name: Caenorhabditis sp. 9) was generated using next-generation sequencing. The mitogenome length is 13,413 bp, containing 12 protein-coding genes (PCGs), 2 ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs) and 2 non-coding regions (NCR). The genome organization and nucleotide composition is very similar to that of the mitogenome of C. elegans and C. briggsae. Mitogenome of C. nigoni shows higher sequence similarity to C. briggsae than to C. elegans, which is consistent with the fact that C. nigoni is a sister species of C. briggsae. However, as in C. elegans, two NCRs present in the mitogenome of C. briggsae are missing in C. nigoni. The mitogenome sequence of C. nigoni plays an important role in further studies of phylogenetics, population genetics and evolutionary genetics in nematode species.

High evolutionary turnover of satellite families in Caenorhabditis.

BACKGROUND: The high density of tandem repeat sequences (satellites) in nematode genomes and the availability of genome sequences from several species in the group offer a unique opportunity to better understand the evolutionary dynamics and the functional role of these sequences. We take advantage of the previously developed SATFIND program to study the satellites in four Caenorhabditis species and investigate these questions. METHODS: The identification and comparison of satellites is carried out in three steps. First we find all the satellites present in each species with the SATFIND program. Each satellite is defined by its length, number of repeats, and repeat sequence. Only satellites with at least ten repeats are considered. In the second step we build satellite families with a newly developed alignment program. Satellite families are defined by a consensus sequence and the number of satellites in the family. Finally we compare the consensus sequence of satellite families in different species. RESULTS: We give a catalog of individual satellites in each species. We have also identified satellite families with a related sequence and compare them in different species. We analyze the turnover of satellites: they increased in size through duplications of fragments of 100-300 bases. It appears that in many cases they have undergone an explosive expansion. In C. elegans we have identified a subset of large satellites that have strong affinity for the centromere protein CENP-A. We have also compared our results with those obtained from other species, including one nematode and three mammals. CONCLUSIONS: Most satellite families found in Caenorhabditis are species-specific; in particular those with long repeats. A subset of these satellites may facilitate the formation of kinetochores in mitosis. Other satellite families in C. elegans are either related to Helitron transposons or to meiotic pairing centers.

Travelling at a slug's pace: possible invertebrate vectors of Caenorhabditis nematodes.

BACKGROUND: How do very small animals with limited long-distance dispersal abilities move between locations, especially if they prefer ephemeral micro-habitats that are only available for short periods of time? The free-living model nematode Caenorhabditis elegans and several congeneric taxa appear to be common in such short-lived environments, for example decomposing fruits or other rotting plant material. Dispersal is usually assumed to depend on animal vectors, yet all current data is based on only a limited number of studies. In our project we performed three comprehensive field surveys on possible invertebrate vectors in North German locations containing populations of C. elegans and two related species, especially C. remanei, and combined these screens with an experimental analysis of persistence in one of the vector taxa. RESULTS: Our field survey revealed that Caenorhabditis nematodes are commonly found in slugs, isopods, and chilopods, but are not present in the remaining taxonomic groups examined. Surprisingly, the nematodes were frequently isolated from the intestines of slugs, even if slugs were not collected in close association with suitable substrates for Caenorhabditis proliferation. This suggests that the nematodes are able to enter the slug intestines and persist for certain periods of time. Our experimental analysis confirmed the ability of C. elegans to invade slug intestines and subsequently be excreted alive with the slug feces, although only for short time periods under laboratory conditions. CONCLUSIONS: We conclude that three invertebrate taxonomic groups represent potential vectors of Caenorhabditis nematodes. The nematodes appear to have evolved specific adaptations to enter and persist in the harsh environment of slug intestines, possibly indicating first steps towards a parasitic life-style.