Environmental radiation exposure at Chornobyl has not systematically affected the genomes or chemical mutagen tolerance phenotypes of local worms.

The 1986 disaster at the Chornobyl Nuclear Power Plant transformed the surrounding region into the most radioactive landscape known on the planet. Whether or not this sudden environmental shift selected for species, or even individuals within a species, that are naturally more resistant to mutagen exposure remains an open question. In this study, we collected, cultured, and cryopreserved 298 wild nematode isolates from areas varying in radioactivity within the Chornobyl Exclusion Zone. We sequenced and assembled genomes de novo for 20 Oscheius tipulae strains, analyzed their genomes for evidence of recent mutation acquisition in the field, and observed no evidence of an association between mutation and radioactivity at the sites of collection. Multigenerational exposure of each of these strains to several chemical mutagens in the lab revealed that strains vary heritably in tolerance to each mutagen, but mutagen tolerance cannot be predicted based on the radiation levels at collection sites, and Chornobyl isolates were not systematically more resistant than strains from undisturbed habitats. In sum, the absence of mutational signatures does not reflect unique capacity for tolerating DNA damage.

Environmental radiation exposure at Chornobyl has not systematically affected the genomes or mutagen tolerance phenotypes of local worms.

The 1986 disaster at the Chornobyl Nuclear Power Plant transformed the surrounding region into the most radioactive landscape known on the planet. Questions remain regarding whether this sudden environmental shift selected for species, or even individuals within a species, that are naturally more resistant to radiation exposure. We collected, cultured, and cryopreserved 298 wild nematodes isolates from areas varying in radioactivity within the Chornobyl Exclusion Zone. We sequenced and assembled genomes de novo for 20 Oschieus tipulae strains, analyzed their genomes for evidence of recent mutation acquisition in the field and saw no evidence of an association between mutation and radiation level at the sites of collection. Multigenerational exposure of each of these strains to several mutagens in the lab revealed that strains vary heritably in tolerance to each mutagen, but mutagen tolerance cannot be predicted based on the radiation levels at collection sites.

Caenorhabditis nematodes colonize ephemeral resource patches in neotropical forests.

Factors shaping the distribution and abundance of species include life-history traits, population structure, and stochastic colonization-extinction dynamics. Field studies of model species groups help reveal the roles of these factors. Species of Caenorhabditis nematodes are highly divergent at the sequence level but exhibit highly conserved morphology, and many of these species live in sympatry on microbe-rich patches of rotten material. Here, we use field experiments and large-scale opportunistic collections to investigate species composition, abundance, and colonization efficiency of Caenorhabditis species in two of the world's best-studied lowland tropical field sites: Barro Colorado Island in Panamá and La Selva in Sarapiquí, Costa Rica. We observed seven species of Caenorhabditis, four of them known only from these collections. We formally describe two species and place them within the Caenorhabditis phylogeny. While these localities contain species from many parts of the phylogeny, both localities were dominated by globally distributed androdiecious species. We found that Caenorhabditis individuals were able to colonize baits accessible only through phoresy and preferentially colonized baits that were in direct contact with the ground. We estimate the number of colonization events per patch to be low.

Natural genetic variation as a tool for discovery in Caenorhabditis nematodes.

Over the last 20 years, studies of Caenorhabditis elegans natural diversity have demonstrated the power of quantitative genetic approaches to reveal the evolutionary, ecological, and genetic factors that shape traits. These studies complement the use of the laboratory-adapted strain N2 and enable additional discoveries not possible using only one genetic background. In this chapter, we describe how to perform quantitative genetic studies in Caenorhabditis, with an emphasis on C. elegans. These approaches use correlations between genotype and phenotype across populations of genetically diverse individuals to discover the genetic causes of phenotypic variation. We present methods that use linkage, near-isogenic lines, association, and bulk-segregant mapping, and we describe the advantages and disadvantages of each approach. The power of C. elegans quantitative genetic mapping is best shown in the ability to connect phenotypic differences to specific genes and variants. We will present methods to narrow genomic regions to candidate genes and then tests to identify the gene or variant involved in a quantitative trait. The same features that make C. elegans a preeminent experimental model animal contribute to its exceptional value as a tool to understand natural phenotypic variation.

Rapid Isolation of Wild Nematodes by Baermann Funnel.

Beyond being robust experimental model organisms, Caenorhabditis elegans and its relatives are also real animals that live in nature. Studies of wild nematodes in their natural environments are valuable for understanding many aspects of biology, including the selective regimes in which distinctive genomic and phenotypic characters evolve, the genetic basis for complex trait variation, and the natural genetic diversity fundamental to all animal populations. This manuscript describes a simple and efficient method for extracting nematodes from their natural substrates, including rotting fruits, flowers, fungi, leaf litter, and soil. The Baermann funnel method, a classical nematology technique, selectively isolates active nematodes from their substrates. Because it recovers nearly all active worms from the sample, the Baermann funnel technique allows for the recovery of rare and slow-growing genotypes that co-occur with abundant and fast-growing genotypes, which might be missed in extraction methods that involve multiple generations of reproduction. The technique is also well suited to addressing metagenetic, population-genetic, and ecological questions. It captures the entire population in a sample simultaneously, allowing an unbiased view of the natural distribution of ages, sexes, and genotypes. The protocol allows for deployment at scale in the field, rapidly converting substrates into worm plates, and the authors have validated it through fieldwork on multiple continents.

The Genome of the Poecilogonous Annelid Streblospio benedicti.

Streblospio benedicti is a common marine annelid that has become an important model for developmental evolution. It is the only known example of poecilogony (where two distinct developmental modes occur within a single species) that is due to a heritable difference in egg size. The dimorphic developmental programs and life-histories exhibited in this species depend on differences within the genome, making it an optimal model for understanding the genomic basis of developmental divergence. Studies using S. benedicti have begun to uncover the genetic and genomic principles that underlie developmental uncoupling, but until now they have been limited by the lack of availability of genomic tools. Here, we present an annotated chromosomal-level genome assembly of S. benedicti generated from a combination of Illumina reads, Nanopore long reads, Chicago and Hi-C chromatin interaction sequencing, and a genetic map from experimental crosses. At 701.4 Mb, the S. benedicti genome is the largest annelid genome to date that has been assembled to chromosomal scaffolds. The complete genome of S. benedicti is valuable for functional genomic analyses of development and evolution, as well as phylogenetic comparison within the annelida and the Lophotrochozoa. Despite having two developmental modes, there is no evidence of genome duplication or substantial gene number expansions. Instead, lineage-specific repeats account for much of the expansion of this genome compared with other annelids.

Baby makes three: Maternal, paternal, and zygotic genetic effects shape larval phenotypic evolution.

The evolutionary potential of a population is shaped by the genetic architecture of its life-history traits. Early-life phenotypes are influenced by both maternal and offspring genotype, and efforts to understand life-history evolution therefore require consideration of the interactions between these separate but correlated genomes. We used a four-generation experimental pedigree to estimate the genetic architecture of early-life phenotypes in a species with dramatic variation in larval size and morphology. In the polychaete annelid Streblospio benedicti, females make either many small eggs that develop into complex larvae that feed in the plankton or few large eggs that develop into benthic juveniles without having to feed as larvae. By isolating the contributions of maternal, paternal, and zygotic genotype to larval traits, we determined that larval anatomical structures are governed by the offspring genotype at a small number of large-effect loci. Larval size is not shaped by the larva's own genotype but instead depends on loci that act in the mother, and at two genomic locations, by loci that act in the father. The overall phenotype of each larva thus depends on three separate genomes, and a population's response to selection on larval traits will reflect the interactions among them.

Balancing selection maintains hyper-divergent haplotypes in Caenorhabditis elegans.

Across diverse taxa, selfing species have evolved independently from outcrossing species thousands of times. The transition from outcrossing to selfing decreases the effective population size, effective recombination rate and heterozygosity within a species. These changes lead to a reduction in genetic diversity, and therefore adaptive potential, by intensifying the effects of random genetic drift and linked selection. Within the nematode genus Caenorhabditis, selfing has evolved at least three times, and all three species, including the model organism Caenorhabditis elegans, show substantially reduced genetic diversity relative to outcrossing species. Selfing and outcrossing Caenorhabditis species are often found in the same niches, but we still do not know how selfing species with limited genetic diversity can adapt to these environments. Here, we examine the whole-genome sequences from 609 wild C. elegans strains isolated worldwide and show that genetic variation is concentrated in punctuated hyper-divergent regions that cover 20% of the C. elegans reference genome. These regions are enriched in environmental response genes that mediate sensory perception, pathogen response and xenobiotic stress response. Population genomic evidence suggests that genetic diversity in these regions has been maintained by long-term balancing selection. Using long-read genome assemblies for 15 wild strains, we show that hyper-divergent haplotypes contain unique sets of genes and show levels of divergence comparable to levels found between Caenorhabditis species that diverged millions of years ago. These results provide an example of how species can avoid the evolutionary dead end associated with selfing.

Gene-level quantitative trait mapping in Caenorhabditis elegans.

The Caenorhabditis elegans multiparental experimental evolution (CeMEE) panel is a collection of genome-sequenced, cryopreserved recombinant inbred lines useful for mapping the evolution and genetic basis of quantitative traits. We have expanded the resource with new lines and new populations, and here report the genotype and haplotype composition of CeMEE version 2, including a large set of putative de novo mutations, and updated additive and epistatic mapping simulations. Additive quantitative trait loci explaining 4% of trait variance are detected with >80% power, and the median detection interval approaches single-gene resolution on the highly recombinant chromosome arms. Although CeMEE populations are derived from a long-term evolution experiment, genetic structure is dominated by variation present in the ancestral population.

Selfing is the safest sex for Caenorhabditis tropicalis.

Mating systems have profound effects on genetic diversity and compatibility. The convergent evolution of self-fertilization in three Caenorhabditis species provides a powerful lens to examine causes and consequences of mating system transitions. Among the selfers, Caenorhabditis tropicalis is the least genetically diverse and most afflicted by outbreeding depression. We generated a chromosomal-scale genome for C. tropicalis and surveyed global diversity. Population structure is very strong, and islands of extreme divergence punctuate a genomic background that is highly homogeneous around the globe. Outbreeding depression in the laboratory is caused largely by multiple Medea-like elements, genetically consistent with maternal toxin/zygotic antidote systems. Loci with Medea activity harbor novel and duplicated genes, and their activity is modified by mito-nuclear background. Segregating Medea elements dramatically reduce fitness, and simulations show that selfing limits their spread. Frequent selfing in C. tropicalis may therefore be a strategy to avoid Medea-mediated outbreeding depression.

The Ancestral Caenorhabditis elegans Cuticle Suppresses rol-1.

Genetic background commonly modifies the effects of mutations. We discovered that worms mutant for the canonical rol-1 gene, identified by Brenner in 1974, do not roll in the genetic background of the wild strain CB4856. Using linkage mapping, association analysis and gene editing, we determined that N2 carries an insertion in the collagen gene col-182 that acts as a recessive enhancer of rol-1 rolling. From population and comparative genomics, we infer the insertion is derived in N2 and related laboratory lines, likely arising during the domestication of Caenorhabditis elegans, and breaking a conserved protein. The ancestral version of col-182 also modifies the phenotypes of four other classical cuticle mutant alleles, and the effects of natural genetic variation on worm shape and locomotion. These results underscore the importance of genetic background and the serendipity of Brenner's choice of strain.

Hybridization promotes asexual reproduction in Caenorhabditis nematodes.

Although most unicellular organisms reproduce asexually, most multicellular eukaryotes are obligately sexual. This implies that there are strong barriers that prevent the origin or maintenance of asexuality arising from an obligately sexual ancestor. By studying rare asexual animal species we can gain a better understanding of the circumstances that facilitate their evolution from a sexual ancestor. Of the known asexual animal species, many originated by hybridization between two ancestral sexual species. The balance hypothesis predicts that genetic incompatibilities between the divergent genomes in hybrids can modify meiosis and facilitate asexual reproduction, but there are few instances where this has been shown. Here we report that hybridizing two sexual Caenorhabditis nematode species (C. nouraguensis females and C. becei males) alters the normal inheritance of the maternal and paternal genomes during the formation of hybrid zygotes. Most offspring of this interspecies cross die during embryogenesis, exhibiting inheritance of a diploid C. nouraguensis maternal genome and incomplete inheritance of C. becei paternal DNA. However, a small fraction of offspring develop into viable adults that can be either fertile or sterile. Fertile offspring are produced asexually by sperm-dependent parthenogenesis (also called gynogenesis or pseudogamy); these progeny inherit a diploid maternal genome but fail to inherit a paternal genome. Sterile offspring are hybrids that inherit both a diploid maternal genome and a haploid paternal genome. Whole-genome sequencing of individual viable worms shows that diploid maternal inheritance in both fertile and sterile offspring results from an altered meiosis in C. nouraguensis oocytes and the inheritance of two randomly selected homologous chromatids. We hypothesize that hybrid incompatibility between C. nouraguensis and C. becei modifies maternal and paternal genome inheritance and indirectly induces gynogenetic reproduction. This system can be used to dissect the molecular mechanisms by which hybrid incompatibilities can facilitate the emergence of asexual reproduction.

Tightly linked antagonistic-effect loci underlie polygenic phenotypic variation in C. elegans.

Recent work has provided strong empirical support for the classic polygenic model for trait variation. Population-based findings suggest that most regions of genome harbor variation affecting most traits. Here, we use the approach of experimental genetics to show that, indeed, most genomic regions carry variants with detectable effects on growth and reproduction in Caenorhabditis elegans populations sensitized by nickel stress. Nine of 15 adjacent intervals on the X chromosome, each encompassing ∼0.001 of the genome, have significant effects when tested individually in near-isogenic lines (NILs). These intervals have effects that are similar in magnitude to those of genome-wide significant loci that we mapped in a panel of recombinant inbred advanced intercross lines (RIAILs). If NIL-like effects were randomly distributed across the genome, the RIAILs would exhibit phenotypic variance that far exceeds the observed variance. However, the NIL intervals are arranged in a pattern that significantly reduces phenotypic variance relative to a random arrangement; adjacent intervals antagonize one another, cancelling each other's effects. Contrary to the expectation of small additive effects, our findings point to large-effect variants whose effects are masked by epistasis or linkage disequilibrium between alleles of opposing effect.

Comparative genomics of 10 new Caenorhabditis species.

The nematode Caenorhabditis elegans has been central to the understanding of metazoan biology. However, C. elegans is but one species among millions and the significance of this important model organism will only be fully revealed if it is placed in a rich evolutionary context. Global sampling efforts have led to the discovery of over 50 putative species from the genus Caenorhabditis, many of which await formal species description. Here, we present species descriptions for 10 new Caenorhabditis species. We also present draft genome sequences for nine of these new species, along with a transcriptome assembly for one. We exploit these whole-genome data to reconstruct the Caenorhabditis phylogeny and use this phylogenetic tree to dissect the evolution of morphology in the genus. We reveal extensive variation in genome size and investigate the molecular processes that underlie this variation. We show unexpected complexity in the evolutionary history of key developmental pathway genes. These new species and the associated genomic resources will be essential in our attempts to understand the evolutionary origins of the C. elegans model.

Decoupled maternal and zygotic genetic effects shape the evolution of development.

Evolutionary transitions from indirect to direct development involve changes in both maternal and zygotic genetic factors, with distinctive population-genetic implications, but empirical data on the genetics of such transitions are lacking. The polychaete Streblospio benedicti provides an opportunity to dissect a major transition in developmental mode using forward genetics. Females in this species produce either small eggs that develop into planktonic larvae or large eggs that develop into benthic juveniles. We identify large-effect loci that act maternally to influence larval size and independent, unlinked large-effect loci that act zygotically to affect discrete aspects of larval morphology. The likely fitness of zygotic alleles depends on their maternal background, creating a positive frequency-dependence that may homogenize local populations. Developmental and population genetics interact to shape larval evolution.

Polygenicity and Epistasis Underlie Fitness-Proximal Traits in the Caenorhabditis elegans Multiparental Experimental Evolution (CeMEE) Panel.

Understanding the genetic basis of complex traits remains a major challenge in biology. Polygenicity, phenotypic plasticity, and epistasis contribute to phenotypic variance in ways that are rarely clear. This uncertainty can be problematic for estimating heritability, for predicting individual phenotypes from genomic data, and for parameterizing models of phenotypic evolution. Here, we report an advanced recombinant inbred line (RIL) quantitative trait locus mapping panel for the hermaphroditic nematode Caenorhabditis elegans, the C. elegans multiparental experimental evolution (CeMEE) panel. The CeMEE panel, comprising 507 RILs at present, was created by hybridization of 16 wild isolates, experimental evolution for 140-190 generations, and inbreeding by selfing for 13-16 generations. The panel contains 22% of single-nucleotide polymorphisms known to segregate in natural populations, and complements existing C. elegans mapping resources by providing fine resolution and high nucleotide diversity across > 95% of the genome. We apply it to study the genetic basis of two fitness components, fertility and hermaphrodite body size at time of reproduction, with high broad-sense heritability in the CeMEE. While simulations show that we should detect common alleles with additive effects as small as 5%, at gene-level resolution, the genetic architectures of these traits do not feature such alleles. We instead find that a significant fraction of trait variance, approaching 40% for fertility, can be explained by sign epistasis with main effects below the detection limit. In congruence, phenotype prediction from genomic similarity, while generally poor ([Formula: see text]), requires modeling epistasis for optimal accuracy, with most variance attributed to the rapidly evolving chromosome arms.

The Genetic Basis of Natural Variation in Caenorhabditis elegans Telomere Length.

Telomeres are involved in the maintenance of chromosomes and the prevention of genome instability. Despite this central importance, significant variation in telomere length has been observed in a variety of organisms. The genetic determinants of telomere-length variation and their effects on organismal fitness are largely unexplored. Here, we describe natural variation in telomere length across the Caenorhabditis elegans species. We identify a large-effect variant that contributes to differences in telomere length. The variant alters the conserved oligonucleotide/oligosaccharide-binding fold of protection of telomeres 2 (POT-2), a homolog of a human telomere-capping shelterin complex subunit. Mutations within this domain likely reduce the ability of POT-2 to bind telomeric DNA, thereby increasing telomere length. We find that telomere-length variation does not correlate with offspring production or longevity in C. elegans wild isolates, suggesting that naturally long telomeres play a limited role in modifying fitness phenotypes in C. elegans.

Fine-Scale Crossover Rate Variation on the Caenorhabditis elegans X Chromosome.

Meiotic recombination creates genotypic diversity within species. Recombination rates vary substantially across taxa, and the distribution of crossovers can differ significantly among populations and between sexes. Crossover locations within species have been found to vary by chromosome and by position within chromosomes, where most crossover events occur in small regions known as recombination hotspots. However, several species appear to lack hotspots despite significant crossover heterogeneity. The nematode Caenorhabditis elegans was previously found to have the least fine-scale variation in crossover distribution among organisms studied to date. It is unclear whether this pattern extends to the X chromosome given its unique compaction through the pachytene stage of meiotic prophase in hermaphrodites. We generated 798 recombinant nested near-isogenic lines (NILs) with crossovers in a 1.41 Mb region on the left arm of the X chromosome to determine if its recombination landscape is similar to that of the autosomes. We find that the fine-scale variation in crossover rate is lower than that of other model species, and is inconsistent with hotspots. The relationship of genomic features to crossover rate is dependent on scale, with GC content, histone modifications, and nucleosome occupancy being negatively associated with crossovers. We also find that the abundances of 4- to 6-bp DNA motifs significantly explain crossover density. These results are consistent with recombination occurring at unevenly distributed sites of open chromatin.

Natural Variation in plep-1 Causes Male-Male Copulatory Behavior in C. elegans.

In sexual species, gametes have to find and recognize one another. Signaling is thus central to sexual reproduction and involves a rapidly evolving interplay of shared and divergent interests [1-4]. Among Caenorhabditis nematodes, three species have evolved self-fertilization, changing the balance of intersexual relations [5]. Males in these androdioecious species are rare, and the evolutionary interests of hermaphrodites dominate. Signaling has shifted accordingly, with females losing behavioral responses to males [6, 7] and males losing competitive abilities [8, 9]. Males in these species also show variable same-sex and autocopulatory mating behaviors [6, 10]. These behaviors could have evolved by relaxed selection on male function, accumulation of sexually antagonistic alleles that benefit hermaphrodites and harm males [5, 11], or neither of these, because androdioecy also reduces the ability of populations to respond to selection [12-14]. We have identified the genetic cause of a male-male mating behavior exhibited by geographically dispersed C. elegans isolates, wherein males mate with and deposit copulatory plugs on one another's excretory pores. We find a single locus of major effect that is explained by segregation of a loss-of-function mutation in an uncharacterized gene, plep-1, expressed in the excretory cell in both sexes. Males homozygous for the plep-1 mutation have excretory pores that are attractive or receptive to copulatory behavior of other males. Excretory pore plugs are injurious and hermaphrodite activity is compromised in plep-1 mutants, so the allele might be unconditionally deleterious, persisting in the population because the species' androdioecious mating system limits the reach of selection.