Genomic diversity landscapes in outcrossing and selfing Caenorhabditis nematodes.

Caenorhabditis nematodes form an excellent model for studying how the mode of reproduction affects genetic diversity, as some species reproduce via outcrossing whereas others can self-fertilize. Currently, chromosome-level patterns of diversity and recombination are only available for self-reproducing Caenorhabditis, making the generality of genomic patterns across the genus unclear given the profound potential influence of reproductive mode. Here we present a whole-genome diversity landscape, coupled with a new genetic map, for the outcrossing nematode C. remanei. We demonstrate that the genomic distribution of recombination in C. remanei, like the model nematode C. elegans, shows high recombination rates on chromosome arms and low rates toward the central regions. Patterns of genetic variation across the genome are also similar between these species, but differ dramatically in scale, being tenfold greater for C. remanei. Historical reconstructions of variation in effective population size over the past million generations echo this difference in polymorphism. Evolutionary simulations demonstrate how selection, recombination, mutation, and selfing shape variation along the genome, and that multiple drivers can produce patterns similar to those observed in natural populations. The results illustrate how genome organization and selection play a crucial role in shaping the genomic pattern of diversity whereas demographic processes scale the level of diversity across the genome as a whole.

Full-genome evolutionary histories of selfing, splitting, and selection in Caenorhabditis.

The nematode Caenorhabditis briggsae is a model for comparative developmental evolution with C. elegans. Worldwide collections of C. briggsae have implicated an intriguing history of divergence among genetic groups separated by latitude, or by restricted geography, that is being exploited to dissect the genetic basis to adaptive evolution and reproductive incompatibility; yet, the genomic scope and timing of population divergence is unclear. We performed high-coverage whole-genome sequencing of 37 wild isolates of the nematode C. briggsae and applied a pairwise sequentially Markovian coalescent (PSMC) model to 703 combinations of genomic haplotypes to draw inferences about population history, the genomic scope of natural selection, and to compare with 40 wild isolates of C. elegans. We estimate that a diaspora of at least six distinct C. briggsae lineages separated from one another approximately 200,000 generations ago, including the "Temperate" and "Tropical" phylogeographic groups that dominate most samples worldwide. Moreover, an ancient population split in its history approximately 2 million generations ago, coupled with only rare gene flow among lineage groups, validates this system as a model for incipient speciation. Low versus high recombination regions of the genome give distinct signatures of population size change through time, indicative of widespread effects of selection on highly linked portions of the genome owing to extreme inbreeding by self-fertilization. Analysis of functional mutations indicates that genomic context, owing to selection that acts on long linkage blocks, is a more important driver of population variation than are the functional attributes of the individually encoded genes.

Comparative genomic analysis of upstream miRNA regulatory motifs in Caenorhabditis.

MicroRNAs (miRNAs) comprise a class of short noncoding RNA molecules that play diverse developmental and physiological roles by controlling mRNA abundance and protein output of the vast majority of transcripts. Despite the importance of miRNAs in regulating gene function, we still lack a complete understanding of how miRNAs themselves are transcriptionally regulated. To fill this gap, we predicted regulatory sequences by searching for abundant short motifs located upstream of miRNAs in eight species of Caenorhabditis nematodes. We identified three conserved motifs across the Caenorhabditis phylogeny that show clear signatures of purifying selection from comparative genomics, patterns of nucleotide changes in motifs of orthologous miRNAs, and correlation between motif incidence and miRNA expression. We then validated our predictions with transgenic green fluorescent protein reporters and site-directed mutagenesis for a subset of motifs located in an enhancer region upstream of let-7 We demonstrate that a CT-dinucleotide motif is sufficient for proper expression of GFP in the seam cells of adult C. elegans, and that two other motifs play incremental roles in combination with the CT-rich motif. Thus, functional tests of sequence motifs identified through analysis of molecular evolutionary signatures provide a powerful path for efficiently characterizing the transcriptional regulation of miRNA genes.

When natural selection gives gene function the cold shoulder.

It is tempting to invoke organismal selection as perpetually optimizing the function of any given gene. However, natural selection can drive genic functional change without improvement of biochemical activity, even to the extinction of gene activity. Detrimental mutations can creep in owing to linkage with other selectively favored loci. Selection can promote functional degradation, irrespective of genetic drift, when adaptation occurs by loss of gene function. Even stabilizing selection on a trait can lead to divergence of the underlying molecular constituents. Selfish genetic elements can also proliferate independent of any functional benefits to the host genome. Here we review the logic and evidence for these diverse processes acting in genome evolution. This collection of distinct evolutionary phenomena - while operating through easily understandable mechanisms - all contribute to the seemingly counterintuitive notion that maintenance or improvement of a gene's biochemical function sometimes do not determine its evolutionary fate.

Ephemeral-habitat colonization and neotropical species richness of Caenorhabditis nematodes.

BACKGROUND: The drivers of species co-existence in local communities are especially enigmatic for assemblages of morphologically cryptic species. Here we characterize the colonization dynamics and abundance of nine species of Caenorhabditis nematodes in neotropical French Guiana, the most speciose known assemblage of this genus, with resource use overlap and notoriously similar external morphology despite deep genomic divergence. METHODS: To characterize the dynamics and specificity of colonization and exploitation of ephemeral resource patches, we conducted manipulative field experiments and the largest sampling effort to date for Caenorhabditis outside of Europe. This effort provides the first in-depth quantitative analysis of substrate specificity for Caenorhabditis in natural, unperturbed habitats. RESULTS: We amassed a total of 626 strain isolates from nine species of Caenorhabditis among 2865 substrate samples. With the two new species described here (C. astrocarya and C. dolens), we estimate that our sampling procedures will discover few additional species of these microbivorous animals in this tropical rainforest system. We demonstrate experimentally that the two most prevalent species (C. nouraguensis and C. tropicalis) rapidly colonize fresh resource patches, whereas at least one rarer species shows specialist micro-habitat fidelity. CONCLUSION: Despite the potential to colonize rapidly, these ephemeral patchy resources of rotting fruits and flowers are likely to often remain uncolonized by Caenorhabditis prior to their complete decay, implying dispersal-limited resource exploitation. We hypothesize that a combination of rapid colonization, high ephemerality of resource patches, and species heterogeneity in degree of specialization on micro-habitats and life histories enables a dynamic co-existence of so many morphologically cryptic species of Caenorhabditis.

No Evidence that MicroRNAs Coevolve with Genes Located in Copy Number Regions.

MicroRNAs (miRNAs) are a widespread class of regulatory noncoding RNAs with key roles in physiology and development, conferring robustness to noise in regulatory networks. Consistent with this buffering function, it was recently suggested that human miRNAs coevolve with genes in copy number regions (copy number variation [CNV] genes) to reduce dosage imbalance. Here, I compare miRNA regulation between CNV and non-CNV genes in four model organisms. miRNA regulation of CNV genes is elevated in human and fly but reduced in nematode and zebrafish. By analyzing 31 human CNV data sets, careful analysis of human and chimpanzee orthologs, resampling genes within species and comparing structural variant types, I show that the apparent coevolution between CNV genes and miRNAs is due to the strong dependency between 3'-untranslated region length and miRNA target prediction. Deciphering the interplay between CNVs and miRNAs will likely require a deeper understanding of how miRNAs are embedded in regulatory circuits.

Specialist versus generalist life histories and nucleotide diversity in Caenorhabditis nematodes.

Species with broad ecological amplitudes with respect to a key focal resource, niche generalists, should maintain larger and more connected populations than niche specialists, leading to the prediction that nucleotide diversity will be lower and more subdivided in specialists relative to their generalist relatives. This logic describes the specialist-generalist variation hypothesis (SGVH). Some outbreeding species of Caenorhabditis nematodes use a variety of invertebrate dispersal vectors and have high molecular diversity. By contrast, Caenorhabditis japonica lives in a strict association and synchronized life cycle with its dispersal host, the shield bug Parastrachia japonensis, itself a diet specialist. Here, we characterize sequence variation for 20 nuclear loci to investigate how C. japonica's life history shapes nucleotide diversity. We find that C. japonica has more than threefold lower polymorphism than other outbreeding Caenorhabditis species, but that local populations are not genetically disconnected. Coupled with its restricted range, we propose that its specialist host association contributes to a smaller effective population size and lower genetic variation than host generalist Caenorhabditis species with outbreeding reproductive modes. A literature survey of diverse organisms provides broader support for the SGVH. These findings encourage further testing of ecological and evolutionary hypotheses with comparative population genetics in Caenorhabditis and other taxa.

Early Cancer Detection in Li-Fraumeni Syndrome with Cell-Free DNA.

People with Li-Fraumeni syndrome (LFS) harbor a germline pathogenic variant in the TP53 tumor suppressor gene, face a near 100% lifetime risk of cancer, and routinely undergo intensive surveillance protocols. Liquid biopsy has become an attractive tool for a range of clinical applications, including early cancer detection. Here, we provide a proof-of-principle for a multimodal liquid biopsy assay that integrates a targeted gene panel, shallow whole-genome, and cell-free methylated DNA immunoprecipitation sequencing for the early detection of cancer in a longitudinal cohort of 89 LFS patients. Multimodal analysis increased our detection rate in patients with an active cancer diagnosis over uni-modal analysis and was able to detect cancer-associated signal(s) in carriers prior to diagnosis with conventional screening (positive predictive value = 67.6%, negative predictive value = 96.5%). Although adoption of liquid biopsy into current surveillance will require further clinical validation, this study provides a framework for individuals with LFS. SIGNIFICANCE: By utilizing an integrated cell-free DNA approach, liquid biopsy shows earlier detection of cancer in patients with LFS compared with current clinical surveillance methods such as imaging. Liquid biopsy provides improved accessibility and sensitivity, complementing current clinical surveillance methods to provide better care for these patients. See related commentary by Latham et al., p. 23. This article is featured in Selected Articles from This Issue, p. 5.

Species richness, distribution and genetic diversity of Caenorhabditis nematodes in a remote tropical rainforest.

BACKGROUND: In stark contrast to the wealth of detail about C. elegans developmental biology and molecular genetics, biologists lack basic data for understanding the abundance and distribution of Caenorhabditis species in natural areas that are unperturbed by human influence. METHODS: Here we report the analysis of dense sampling from a small, remote site in the Amazonian rain forest of the Nouragues Natural Reserve in French Guiana. RESULTS: Sampling of rotting fruits and flowers revealed proliferating populations of Caenorhabditis, with up to three different species co-occurring within a single substrate sample, indicating remarkable overlap of local microhabitats. We isolated six species, representing the highest local species richness for Caenorhabditis encountered to date, including both tropically cosmopolitan and geographically restricted species not previously isolated elsewhere. We also documented the structure of within-species molecular diversity at multiple spatial scales, focusing on 57 C. briggsae isolates from French Guiana. Two distinct genetic subgroups co-occur even within a single fruit. However, the structure of C. briggsae population genetic diversity in French Guiana does not result from strong local patterning but instead presents a microcosm of global patterns of differentiation. We further integrate our observations with new data from nearly 50 additional recently collected C. briggsae isolates from both tropical and temperate regions of the world to re-evaluate local and global patterns of intraspecific diversity, providing the most comprehensive analysis to date for C. briggsae population structure across multiple spatial scales. CONCLUSIONS: The abundance and species richness of Caenorhabditis nematodes is high in a Neotropical rainforest habitat that is subject to minimal human interference. Microhabitat preferences overlap for different local species, although global distributions include both cosmopolitan and geographically restricted groups. Local samples for the cosmopolitan C. briggsae mirror its pan-tropical patterns of intraspecific polymorphism. It remains an important challenge to decipher what drives Caenorhabditis distributions and diversity within and between species.

Pleiotropic constraints, expression level, and the evolution of miRNA sequences.

Post-transcriptional gene regulation mediated by microRNAs (miRNAs) plays critical roles during development by modulating gene expression and conferring robustness to stochastic errors. Phylogenetic analyses suggest that miRNA acquisition could play a role in phenotypic innovation. Moreover, miRNA-induced regulation strongly impacts genome evolution, increasing selective constraints on 3'UTRs, protein sequences, and expression level divergence. Thus, it is essential to understand the factors governing sequence evolution for this important class of regulatory molecules. Investigation of the patterns of molecular evolution at miRNA loci have been limited in Caenorhabditis elegans because of the lack of a close outgroup. Instead, I used Caenorhabditis briggsae as the focus point of this study because of its close relationship to Caenorhabditis sp. 9. I also corroborated the patterns of sequence evolution in Caenorhabditis using published orthologous relationships among miRNAs in Drosophila. In nematodes and in flies, miRNA sequence divergence is not influenced by the genomic neighborhood (i.e., intronic or intergenic) but is nevertheless affected by the genomic context because X-linked miRNAs evolve faster than autosomal miRNAs. However, this effect of chromosomal linkage can be explained by differential expression levels rather than a fast-X effect. The results presented here support a universal negative relationship between rates of molecular evolution and expression level, and suggest that mutations in highly expressed miRNAs are more likely to be deleterious because they potentially affect a larger number of target genes. Finally, I show that many single family member miRNAs evolve faster than miRNAs from multigene families and have limited functional scope, suggesting that they are not strongly integrated in gene regulatory networks.

Molecular hyperdiversity and evolution in very large populations.

The genomic density of sequence polymorphisms critically affects the sensitivity of inferences about ongoing sequence evolution, function and demographic history. Most animal and plant genomes have relatively low densities of polymorphisms, but some species are hyperdiverse with neutral nucleotide heterozygosity exceeding 5%. Eukaryotes with extremely large populations, mimicking bacterial and viral populations, present novel opportunities for studying molecular evolution in sexually reproducing taxa with complex development. In particular, hyperdiverse species can help answer controversial questions about the evolution of genome complexity, the limits of natural selection, modes of adaptation and subtleties of the mutation process. However, such systems have some inherent complications and here we identify topics in need of theoretical developments. Close relatives of the model organisms Caenorhabditis elegans and Drosophila melanogaster provide known examples of hyperdiverse eukaryotes, encouraging functional dissection of resulting molecular evolutionary patterns. We recommend how best to exploit hyperdiverse populations for analysis, for example, in quantifying the impact of noncrossover recombination in genomes and for determining the identity and micro-evolutionary selective pressures on noncoding regulatory elements.

A polygenic two-hit hypothesis for prostate cancer.

Prostate cancer is one of the most heritable cancers. Hundreds of germline polymorphisms have been linked to prostate cancer diagnosis and prognosis. Polygenic risk scores can predict genetic risk of a prostate cancer diagnosis. Although these scores inform the probability of developing a tumor, it remains unknown how germline risk influences the tumor molecular evolution. We cultivated a cohort of 1250 localized European-descent patients with germline and somatic DNA profiling. Men of European descent with higher genetic risk were diagnosed earlier and had less genomic instability and fewer driver genes mutated. Higher genetic risk was associated with better outcome. These data imply a polygenic "two-hit" model where germline risk reduces the number of somatic alterations required for tumorigenesis. These findings support further clinical studies of polygenic risk scores as inexpensive and minimally invasive adjuncts to standard risk stratification. Further studies are required to interrogate generalizability to more ancestrally and clinically diverse populations.

Rapid sequence evolution of transcription factors controlling neuron differentiation in Caenorhabditis.

Whether phenotypic evolution proceeds predominantly through changes in regulatory sequences is a controversial issue in evolutionary genetics. Ample evidence indicates that the evolution of gene regulatory networks via changes in cis-regulatory sequences is an important determinant of phenotypic diversity. However, recent experimental work suggests that the role of transcription factor (TF) divergence in developmental evolution may be underestimated. In order to help understand what levels of constraints are acting on the coding sequence of developmental regulatory genes, evolutionary rates were investigated among 48 TFs required for neuronal development in Caenorhabditis elegans. Allelic variation was then sampled for 28 of these genes within a population of the related species Caenorhabditis remanei. Neuronal TFs are more divergent, both within and between species, than structural genes. TFs affecting different neuronal classes are under different levels of selective constraints. The regulatory genes controlling the differentiation of chemosensory neurons evolve particularly fast and exhibit higher levels of within- and between-species nucleotide variation than TFs required for the development of several neuronal classes and TFs required for motorneuron differentiation. The TFs affecting chemosensory neuron development are also more divergent than chemosensory genes expressed in the neurons they differentiate. These results illustrate that TFs are not as highly constrained as commonly thought and suggest that the role of divergence in developmental regulatory genes during the evolution of gene regulatory networks requires further attention.

High nucleotide divergence in developmental regulatory genes contrasts with the structural elements of olfactory pathways in caenorhabditis.

Almost all organismal function is controlled by pathways composed of interacting genetic components. The relationship between pathway structure and the evolution of individual pathway components is not completely understood. For the nematode Caenorhabditis elegans, chemosensory pathways regulate critical aspects of an individual's life history and development. To help understand how olfaction evolves in Caenorhabditis and to examine patterns of gene evolution within transduction pathways in general, we analyzed nucleotide variation within and between species across two well-characterized olfactory pathways, including regulatory genes controlling the fate of the cells in which the pathways are expressed. In agreement with previous studies, we found much higher levels of polymorphism within C. remanei than within the related species C. elegans and C. briggsae. There are significant differences in the rates of nucleotide evolution for genes across the two pathways but no particular association between evolutionary rate and gene position, suggesting that the evolution of functional pathways must be considered within the context of broader gene network structure. However, developmental regulatory genes show both higher levels of divergence and polymorphism than the structural genes of the pathway. These results show that, contrary to the emerging paradigm in the evolution of development, important structural changes can accumulate in transcription factors.

Evolution of developmental regulation in the vertebrate FgfD subfamily.

Fibroblast growth factors (Fgfs) encode small signaling proteins that help regulate embryo patterning. Fgfs fall into seven families, including FgfD. Nonvertebrate chordates have a single FgfD gene; mammals have three (Fgf8, Fgf17, and Fgf18); and teleosts have six (fgf8a, fgf8b, fgf17, fgf18a, fgf18b, and fgf24). What are the evolutionary processes that led to the structural duplication and functional diversification of FgfD genes during vertebrate phylogeny? To study this question, we investigated conserved syntenies, patterns of gene expression, and the distribution of conserved noncoding elements (CNEs) in FgfD genes of stickleback and zebrafish, and compared them with data from cephalochordates, urochordates, and mammals. Genomic analysis suggests that Fgf8, Fgf17, Fgf18, and Fgf24 arose in two rounds of whole genome duplication at the base of the vertebrate radiation; that fgf8 and fgf18 duplications occurred at the base of the teleost radiation; and that Fgf24 is an ohnolog that was lost in the mammalian lineage. Expression analysis suggests that ancestral subfunctions partitioned between gene duplicates and points to the evolution of novel expression domains. Analysis of CNEs, at least some of which are candidate regulatory elements, suggests that ancestral CNEs partitioned between gene duplicates. These results help explain the evolutionary pathways by which the developmentally important family of FgfD molecules arose and the deduced principles that guided FgfD evolution are likely applicable to the evolution of developmental regulation in many vertebrate multigene families.

Hitting two birds with one stone: The unforeseen consequences of nested gene knockouts in Caenorhabditis elegans.

Nested genes represent an intriguing form of non-random genomic organization in which the boundaries of one gene are fully contained within another, longer host gene. The C. elegans genome contains over 10,000 nested genes, 92% of which are ncRNAs, which occur inside 16% of the protein coding gene complement. Host genes are longer than non-host coding genes, owing to their longer and more numerous introns. Indel alleles are available for nearly all of these host genes that simultaneously alter the nested gene, raising the possibility of nested gene disruption contributing to phenotypes that might be attributed to the host gene. Such dual-knockouts could represent a source of misinterpretation about host gene function. Dual-knockouts might also provide a novel source of synthetic phenotypes that reveal the functional effects of ncRNA genes, whereby the host gene disruption acts as a perturbed genetic background to help unmask ncRNA phenotypes.

Microevolution of nematode miRNAs reveals diverse modes of selection.

Micro-RNA (miRNA) genes encode abundant small regulatory RNAs that play key roles during development and in homeostasis by fine tuning and buffering gene expression. This layer of regulatory control over transcriptional networks is preserved by selection across deep evolutionary time, yet selection pressures on individual miRNA genes in contemporary populations remain poorly characterized in any organism. Here, we quantify nucleotide variability for 129 miRNAs in the genome of the nematode Caenorhabditis remanei to understand the microevolution of this important class of regulatory genes. Our analysis of three population samples and C. remanei's sister species revealed ongoing natural selection that constrains evolution of all sequence domains within miRNA hairpins. We also show that new miRNAs evolve faster than older miRNAs but that selection nevertheless favors their persistence. Despite the ongoing importance of purging of new mutations, we discover a trove of >400 natural miRNA sequence variants that include single nucleotide polymorphisms in seed motifs, indels that ablate miRNA functional domains, and origination of new miRNAs by duplication. Moreover, we demonstrate substantial nucleotide divergence of pre-miRNA hairpin alleles between populations and sister species. These findings from the first global survey of miRNA microevolution in Caenorhabditis support the idea that changes in gene expression, mediated through divergence in miRNA regulation, can contribute to phenotypic novelty and adaptation to specific environments in the present day as well as the distant past.

A recent global selective sweep on the age-1 phosphatidylinositol 3-OH kinase regulator of the insulin-like signaling pathway within Caenorhabditis remanei.

The discovery that genetic pathways can be manipulated to extend lifespan has revolutionized our understanding of aging, yet their function within natural populations remains poorly characterized. In particular, evolutionary theories of aging predict tradeoffs in resource investment toward somatic maintenance vs. reproductive output that should impose strong natural selection on genetic components that influence this balance. To explore such selective pressure at the molecular level, we examine population genetic variation in the insulin-like signaling pathway of the nematode Caenorhabditis remanei. We document a recent global selective sweep on the phosphoinositide-3-kinase pathway regulator, age-1, the first life-extension gene to have been identified. In particular, we find that age-1 has 5-20 times less genetic variation than any other insulin-like signaling pathway components and that evolutionary signatures of selection center on the age-1 locus within its genomic environment. These results demonstrate that critical components of aging-related pathways can be subject to shifting patterns of strong selection, as predicted by theory. This highly polymorphic outcrossing species offers high-resolution, population-level analyses of molecular variation as a complement to functional genetic studies within the self-reproducing C. elegans model system.

Fine-scale signatures of molecular evolution reconcile models of indel-associated mutation.

Genomic structural alterations that vary within species, known as large copy number variants, represent an unanticipated and abundant source of genetic diversity that associates with variation in gene expression and susceptibility to disease. Even short insertions and deletions (indels) can exert important effects on genomes by locally increasing the mutation rate, with multiple mechanisms proposed to account for this pattern. To better understand how indels promote genome evolution, we demonstrate that the single nucleotide mutation rate is elevated in the vicinity of indels, with a resolution of tens of base pairs, for the two closely related nematode species Caenorhabditis remanei and C. sp. 23. In addition to indels being clustered with single nucleotide polymorphisms and fixed differences, we also show that transversion mutations are enriched in sequences that flank indels and that many indels associate with sequence repeats. These observations are compatible with a model that reconciles previously proposed mechanisms of indel-associated mutagenesis, implicating repeat sequences as a common driver of indel errors, which then recruit error-prone polymerases during DNA repair, resulting in a locally elevated single nucleotide mutation rate. The striking influence of indel variants on the molecular evolution of flanking sequences strengthens the emerging general view that mutations can induce further mutations.

Outbreeding depression with low genetic variation in selfing Caenorhabditis nematodes.

Theory and empirical study produce clear links between mating system evolution and inbreeding depression. The connections between mating systems and outbreeding depression, whereby fitness is reduced in crosses of less related individuals, however, are less well defined. Here we investigate inbreeding and outbreeding depression in self-fertile androdioecious nematodes, focusing on Caenorhabditis sp. 11. We quantify nucleotide polymorphism for nine nuclear loci for strains throughout its tropical range, and find some evidence of genetic differentiation despite the lowest sequence diversity observed in this genus. Controlled crosses between strains from geographically separated regions show strong outbreeding depression, with reproductive output of F1s reduced by 36% on average. Outbreeding depression is therefore common in self-fertilizing Caenorhabditis species, each of which evolved androdioecious selfing hermaphroditism independently, but appears strongest in C. sp. 11. Moreover, the poor mating efficiency of androdioecious males extends to C. sp. 11. We propose that self-fertilization is a key driver of outbreeding depression, but that it need not evolve as a direct result of local adaptation per se. Our verbal model of this process highlights the need for formal theory, and C. sp. 11 provides a convenient system for testing the genetic mechanisms that cause outbreeding depression, negative epistasis, and incipient speciation.