Hakuna Nematoda: genetic and phenotypic diversity in African isolates of Caenorhabditis elegans and C. briggsae

Caenorhabditis elegans and C. briggsae have many parallels in terms of morphology, life history and breeding system. Both species also share similar low levels of molecular diversity, although the global sampling of natural populations has been limited and geographically biased. In this study, we describe the first cultured isolates of C. elegans and C. briggsae from sub-Saharan Africa. We characterize these samples for patterns of nucleotide polymorphism and vulva precursor cell lineage, and conduct a series of hybrid crosses in C. briggsae to test for genetic incompatibilities. The distribution of genetic diversity confirms a lack of geographic structure to C. elegans sequences but shows genetic differentiation of C. briggsae into three distinct clades that may correspond to three latitudinal ranges. Despite low levels of molecular diversity, we find considerable variation in cell division frequency in African C. elegans for the P3.p vulva precursor cell, and in African C. briggsae for P4.p, a variation that was not previously observed in this species. Hybrid crosses did not reveal major incompatibilities between C. briggsae strains from Africa and elsewhere, and there was some evidence of inbreeding depression. These new African isolates suggest that important ecological factors may be shaping the patterns of diversity in C. briggsae, and that despite many similarities between C. elegans and C. briggsae, there may be more subtle differences in their natural histories than previously appreciated.     

Levels of DNA polymorphism vary with mating system in the nematode genus caenorhabditis

Self-fertilizing species often harbor less genetic variation than cross-fertilizing species, and at least four different models have been proposed to explain this trend. To investigate further the relationship between mating system and genetic variation, levels of DNA sequence polymorphism were compared among three closely related species in the genus Caenorhabditis: two self-fertilizing species, Caenorhabditis elegans and C. briggsae, and one cross-fertilizing species, C. remanei. As expected, estimates of silent site nucleotide diversity were lower in the two self-fertilizing species. For the mitochondrial genome, diversity in the selfing species averaged 42% of diversity in C. remanei. Interestingly, the reduction in genetic variation was much greater for the nuclear than for the mitochondrial genome. For two nuclear genes, diversity in the selfing species averaged 6 and 13% of diversity in C. remanei. We argue that either population bottlenecks or the repeated action of natural selection, coupled with high levels of selfing, are likely to explain the observed reductions in species-wide genetic diversity.     

High nucleotide polymorphism and rapid decay of linkage disequilibrium in wild populations of Caenorhabditis remanei

The common ancestor of the self-fertilizing nematodes Caenorhabditis elegans and C. briggsae must have reproduced by obligate outcrossing, like most species in this genus. However, we have only a limited understanding about how genetic variation is patterned in such male-female (gonochoristic) Caenorhabditis species. Here, we report results from surveying nucleotide variation of six nuclear loci in a broad geographic sample of wild isolates of the gonochoristic C. remanei. We find high levels of diversity in this species, with silent-site diversity averaging 4.7%, implying an effective population size close to 1 million. Additionally, the pattern of polymorphisms reveals little evidence for population structure or deviation from neutral expectations, suggesting that the sampled C. remanei populations approximate panmixis and demographic equilibrium. Combined with the observation that linkage disequilibrium between pairs of polymorphic sites decays rapidly with distance, this suggests that C. remanei will provide an excellent system for identifying the genetic targets of natural selection from deviant patterns of polymorphism and linkage disequilibrium. The patterns revealed in this obligately outcrossing species may provide a useful model of the evolutionary circumstances in C. elegans' gonochoristic progenitor. This will be especially important if self-fertilization evolved recently in C. elegans history, because most of the evolutionary time separating C. elegans from its known relatives would have occurred in a state of obligate outcrossing.     

Comparative genomics and adaptive selection of the ATP-binding-cassette gene family in caenorhabditis species

ABC transporters constitute one of the largest gene families in all species. They are mostly involved in transport of substrates across membranes. We have previously demonstrated that the Caenorhabditis elegans ABC family shows poor one-to-one gene orthology with other distant model organisms. To address the evolution dynamics of this gene family among closely related species, we carried out a comparative analysis of the ABC family among the three nematode species C. elegans, C. briggsae, and C. remanei. In contrast to the previous observations, the majority of ABC genes in the three species were found in orthologous trios, including many tandemly duplicated ABC genes, indicating that the gene duplication took place before speciation. Species-specific expansions of ABC members are rare and mostly observed in subfamilies A and B. C. briggsae and C. remanei orthologous ABC genes tend to cluster on trees, with those of C. elegans as an outgroup, consistent with their proposed species phylogeny. Comparison of intron/exon structures of the highly conserved ABCE subfamily members also indicates a closer relationship between C. briggsae and C. remanei than between either of these species and C. elegans. A comparison between insect and mammalian species indicates lineage-specific duplications or deletions of ABC genes, while the family size remains relatively constant. Sites undergoing positive selection within subfamily D, which are implicated in very-long-chain fatty acid transport, were identified. The evolution of these sites might be driven by the changes in food source with time.     

Regulatory elements required for development of caenorhabditis elegans hermaphrodites are conserved in the tra-2 homologue of C. remanei, a male/female sister species

The Caenorhabditis elegans hermaphrodite is essentially a female that produces sperm. In C. elegans, tra-2 promotes female fates and must be repressed to achieve hermaphrodite spermatogenesis. In an effort to learn how mating systems evolve, we have cloned tra-2 from C. remanei, the closest gonochoristic relative of C. elegans. We found its structure to be similar to that of Ce-tra-2 but its sequence to be divergent. RNA interference demonstrates that Cr-tra-2 promotes female fates. Two sites of tra-2 regulation are required for the onset of hermaphrodite spermatogenesis in C. elegans. One, the MX region of TRA-2, is as well conserved in C. remanei as it is in C. briggsae (another male/hermaphrodite species), suggesting that this control is not unique to hermaphrodites. Another, the DRE/TGE element of the tra-2 3' UTR, was not detected by sequence analysis. However, gel-shift assays demonstrate that a factor in C. remanei can bind specifically to the Cr-tra-2 3' UTR, suggesting that this translational control is also conserved. We propose that both controls are general and do not constitute a novel "switch" that enables sexual mosaicism in hermaphrodites. However, subtle quantitative or qualitative differences in their employment may underlie differences in mating system seen in Caenorhabditis.     

Patterns of nucleotide polymorphism distinguish temperate and tropical wild isolates of Caenorhabditis briggsae

Caenorhabditis briggsae provides a natural comparison species for the model nematode C. elegans, given their similar morphology, life history, and hermaphroditic mode of reproduction. Despite C. briggsae boasting a published genome sequence and establishing Caenorhabditis as a model genus for genetics and development, little is known about genetic variation across the geographic range of this species. In this study, we greatly expand the collection of natural isolates and characterize patterns of nucleotide variation for six loci in 63 strains from three continents. The pattern of polymorphisms reveals differentiation between C. briggsae strains found in temperate localities in the northern hemisphere from those sampled near the Tropic of Cancer, with diversity within the tropical region comparable to what is found for C. elegans in Europe. As in C. elegans, linkage disequilibrium is pervasive, although recombination is evident among some variant sites, indicating that outcrossing has occurred at a low rate in the history of the sample. In contrast to C. elegans, temperate regions harbor extremely little variation, perhaps reflecting colonization and recent expansion of C. briggsae into northern latitudes. We discuss these findings in relation to their implications for selection, demographic history, and the persistence of self-fertilization.     

Conservation of glp-1 regulation and function in nematodes

The Caenorhabditis elegans (Ce) glp-1 gene encodes a Notch-like receptor. We have cloned glp-1 from C. briggsae (Cb) and C. remanei (Cr), two Caenorhabditis species that have diverged from C. elegans by roughly 20-40 million years. By sequence analysis, we find that the Cb-GLP-1 and Cr-GLP-1 proteins have retained the same motif architecture as Ce-GLP-1, including number of domains. In addition, two regions (CC-linker and regions flanking the ANK repeats) are as highly conserved as regions previously recognized as essential for signaling (e.g., ANK repeats). Phylogenetic analysis of glp-1 sequences suggests a C. briggsae/C. remanei clade with C. elegans as a sister taxon. Using RNAi to test biological functions, we find that Ce-glp-1, Cb-glp-1, and Cr-glp-1 are all required for proliferation of germline stem cells and for specifying blastomere fates in the embryo. In addition, certain biological roles of Cb-glp-1, e.g., in the vulva, have diverged from those of Ce-glp-1 and Cr-glp-1, suggesting a change in either regulation or function of the Cb-glp-1 gene during evolution. Finally, the regulation of glp-1 mRNA, previously analyzed for Ce-glp-1, is conserved in Cb-glp-1, and we identify conserved 3' UTR sequences that may serve as regulatory elements.     

Oscheius tipulae, a widespread hermaphroditic soil nematode, displays a higher genetic diversity and geographical structure than Caenorhabditis elegans

The nematode Oscheius tipulae belongs to the same family (Rhabditidae) as the model species Caenorhabditis elegans . Both species reproduce through self-fertilizing hermaphrodites and facultative males. Recent studies have shown that the self-fertile C. elegans and C. briggsae displayed a 20-fold lower genetic diversity than the male–female species C. remanei . Several explanations have been put forward to account for this difference, including their mode of reproduction and dynamic population structure. Here, we present the results of extensive worldwide sampling of O. tipulae , which we previously used as a laboratory organism for developmental genetics. We found that O. tipulae is much more widespread and common in soil throughout the world than Caenorhabditis species. We analysed 63 O. tipulae isolates from several continents using amplified fragment length polymorphism (AFLP). We found that O. tipulae harbours a 5-fold higher genetic diversity than C. elegans and C. briggsae . As in C. elegans , a high proportion of this diversity was found locally. Yet, we detected significant geographical differentiation, both at the worldwide scale with a latitudinal structure and between three localities in France. In summary, O. tipulae exhibited significantly higher levels of genetic diversity and large-scale geographical structure than C. elegans , despite their shared mode of reproduction. This species difference in genetic diversity may be explained by a number of other differences, such as population size, distribution, migration and dynamics. Due to its widespread occurrence and relatively high genetic diversity, O. tipulae may be a promising study species for evolutionary studies.     

Cryptic quantitative evolution of the vulva intercellular signaling network in Caenorhabditis

BACKGROUND: The Caenorhabditis vulva is formed from a row of Pn.p precursor cells, which adopt a spatial cell-fate pattern-3 degrees 3 degrees 2 degrees 1 degrees 2 degrees 3 degrees -centered on the gonadal anchor cell. This pattern is robustly specified by an intercellular signaling network including EGF/Ras induction from the anchor cell and Delta/Notch signaling between the precursor cells. It is unknown how the roles and quantitative contributions of these signaling pathways have evolved in closely related Caenorhabditis species. RESULTS: Cryptic evolution in the network is uncovered by quantification of cell-fate-pattern frequencies obtained after displacement of the system out of its normal range, either by anchor-cell ablations or through LIN-3/EGF overexpression. Silent evolution in the Caenorhabditis genus covers a large neutral space of cell-fate patterns. Direct induction of the 1 degrees fate as in C. elegans appeared within the genus. C. briggsae displays a graded induction of 1 degrees and 2 degrees fates, with 1 degrees fate induction requiring a longer time than in C. elegans, and a reduced lateral inhibition of adjacent 1 degrees fates. C. remanei displays a strong lateral induction of 2 degrees fates relative to vulval-fate activation in the central cell. This evolution in cell-fate pattern space can be experimentally reconstituted by mild variations of Ras, Wnt, and Notch pathway activities in C. elegans and C. briggsae. CONCLUSIONS: Quantitative evolution in the roles of graded induction by LIN-3/EGF and Notch signaling is demonstrated for the Caenorhabditis vulva signaling network. This evolutionary system biology approach provides a quantitative view of the variational properties of this biological system.     

Diversity in mating behavior of hermaphroditic and male-female Caenorhabditis nematodes

In this study, we addressed why Caenorhabditis elegans males are inefficient at fertilizing their hermaphrodites. During copulation, hermaphrodites generally move away from males before they become impregnated. C. elegans hermaphrodites reproduce by internal self-fertilization, so that copulation with males is not required for species propagation. The hermaphroditic mode of reproduction could potentially relax selection for genes that optimize male mating behavior. We examined males from hermaphroditic and gonochoristic (male-female copulation) Caenorhabditis species to determine if they use different sensory and motor mechanisms to control their mating behavior. Instead, we found through laser ablation analysis and behavioral observations that hermaphroditic C. briggsae and gonochoristic C. remanei and Caenorhabditis species 4, PB2801 males produce a factor that immobilizes females during copulation. This factor also stimulates the vulval slit to widen, so that the male copulatory spicules can easily insert. C. elegans and C. briggsae hermaphrodites are not affected by this factor. We suggest that sensory and motor execution of mating behavior have not significantly changed among males of different Caenorhabditis species; however, during the evolution of internal self-fertilization, hermaphrodites have lost the ability to respond to the male soporific-inducing factor.     

Haldane's rule by sexual transformation in Caenorhabditis

Haldane's rule in C. briggsae x C. remanei broods was caused by sexual transformation; XX and XO hybrids were female. C. briggsae and C. remanei variants that partially suppress hybrid sexual transformation were identified. Effects of variant strains were cumulative. Hence, aberrant sex determination is a reproductive isolation mechanism in Caenorhabditis.     

A comparative genomic analysis of the small heat shock proteins in Caenorhabditis elegans and briggsae

The small heat shock proteins (sHSPs) are a ubiquitous family of molecular chaperones. We have identified 18 sHSPs in the Caenorhabditis elegans genome and 20 sHSPs in the Caenorhabditis briggsae genome. Analysis of phylogenetic relationships and evolutionary dynamics of the sHSPs in these two genomes reveals a very complex pattern of evolution. The sHSPs in C. elegans and C. briggsae do not display clear orthologous relationships with other invertebrate sHSPs. But many sHSPs in C. elegans have orthologs in C. briggsae. One group of sHSPs, the HSP16s, has a very unusual evolutionary history. Although there are a number of HSP16s in both the C. elegans and C. briggsae genomes, none of the HSP16s display orthologous relationships across these two species. The HSP16s have an unusual gene pair structure and a complex evolutionary history shaped by gene duplication, gene conversion, and purifying selection. We found no evidence of recent positive selection acting on any of the sHSPs in C. elegans or in C. briggsae. There is also no evidence of functional divergence within the pairs of orthologous C. elegans and C. briggsae sHSPs. However, the evolutionary patterns do suggest that functional divergence has occurred between the sHSPs in C. elegans and C. briggsae and the sHSPs in more distantly related invertebrates.     

Genetic analysis of dauer formation in Caenorhabditis briggsae

Molecular changes that underlie evolutionary changes in behavior and physiology are not well understood. Dauer formation in Caenorhabditis elegans is a temperature-sensitive process controlled through a network of signaling pathways associated with sensory neurons and is potentially an excellent system in which to investigate molecular changes in neuronal function during evolution. To begin to investigate the evolution of dauer formation in the genus Caenorhabditis at the molecular level, we isolated dauer-formation mutations in C. briggsae, a species closely related to the model organism C. elegans. We identified mutations in orthologs of C. elegans genes daf-2 (insulin receptor), daf-3 (Smad), and daf-4 (TGF-beta type 2 receptor), as well as genes required for formation of sensory cilia. Phenotypic analyses revealed that functions of these genes are conserved between C. elegans and C. briggsae. Analysis of C. briggsae mutations also revealed a significant difference between the two species in their responses to high temperatures (>26 degrees). C. elegans is strongly induced to form dauers at temperatures above 26 degrees, near the upper limit for growth of C. elegans. In contrast, C. briggsae, which is capable of growth at higher temperatures than C. elegans, lacks this response.     

Comparative genetics of sex determination: masculinizing mutations in Caenorhabditis briggsae

The nematodes Caenorhabditis elegans and C. briggsae independently evolved self-fertile hermaphroditism from gonochoristic ancestors. C. briggsae has variably divergent orthologs of nearly all genes in the C. elegans sex determination pathway. Their functional characterization has generally relied on reverse genetic approaches, such as RNA interference and cross-species transgene rescue and more recently on deletion mutations. We have taken an unbiased forward mutagenesis approach to isolating zygotic mutations that masculinize all tissues of C. briggsae hermaphrodites. The screens identified loss-of-function mutations in the C. briggsae orthologs of tra-1, tra-2, and tra-3. The somatic and germline phenotypes of these mutations are largely identical to those of their C. elegans homologs, including the poorly understood germline feminization of tra-1(lf) males. This overall conservation of Cb-tra phenotypes is in contrast to the fem genes, with which they directly interact and which are significantly divergent in germline function. In addition, we show that in both C. briggsae and C. elegans large C-terminal truncations of TRA-1 that retain the DNA-binding domain affect sex determination more strongly than somatic gonad development. Beyond these immediate results, this collection of mutations provides an essential foundation for further comparative genetic analysis of the Caenorhabditis sex determination pathway.     

The egg surface LDL receptor repeat-containing proteins EGG-1 and EGG-2 are required for fertilization in Caenorhabditis elegans

The molecular machinery that mediates sperm-egg interactions at fertilization is largely unknown. We identify two partially redundant egg surface LDL receptor repeat-containing proteins (EGG-1 and EGG-2) that are required for Caenorhabditis elegans fertility in hermaphrodites, but not males. Wild-type sperm cannot enter the morphologically normal oocytes produced by hermaphrodites that lack egg-1 and egg-2 function despite direct gamete contact. Furthermore, we find that levels of meiotic maturation/ovulation and sperm migratory behavior are altered in egg-1 mutants. These observations suggest an unexpected regulatory link between fertilization and other events necessary for reproductive success. egg-1 and egg-2 are the result of a gene duplication in the nematode lineage leading to C. elegans. The two closely related species C. briggsae and C. remanei encode only a single egg-1/egg-2 homolog that is required for hermaphrodite/female fertility. In addition to being the first identified egg components of the nematode fertilization machinery, the egg-1 and egg-2 gene duplication could be vital with regards to maximizing C. elegans fecundity and understanding the evolutionary differentiation of molecular function and speciation.     

An alpha-tubulin mutant demonstrates distinguishable functions among the spindle assembly checkpoint genes in Saccharomyces cerevisiae

The free-living nematode worm Caenorhabditis elegans reproduces primarily as a self-fertilizing hermaphrodite, yet males are maintained in wild-type populations at low frequency. To determine the role of males in C. elegans, we develop a mathematical model for the genetic system of hermaphrodites that can either self-fertilize or be fertilized by males and we perform laboratory observations and experiments on both C. elegans and a related dioecious species C. remanei. We show that the mating efficiency of C. elegans is poor compared to a dioecious species and that C. elegans males are more attracted to C. remanei females than they are to their conspecific hermaphrodites. We postulate that a genetic mutation occurred during the evolution of C. elegans hermaphrodites, resulting in the loss of an attracting sex pheromone present in the ancestor of both C. elegans and C. remanei. Our findings suggest that males are maintained in C. elegans because of the particular genetic system inherited from its dioecious ancestor and because of nonadaptive spontaneous nondisjunction of sex chromosomes, which occurs during meiosis in the hermaphrodite. A theoretical argument shows that the low frequency of male mating observed in C. elegans can support male-specific genes against mutational degeneration. This results in the continuing presence of functional males in a 99.9% hermaphroditic species in which outcrossing is disadvantageous to hermaphrodites.     

Conservation of gene organization and trans-splicing in the glyceraldehyde-3-phosphate dehydrogenase-encoding genes of Caenorhabditis briggsae.

The genes encoding body-wall-specific glyceraldehyde-3-phosphate dehydrogenase from Caenorhabditis briggsae were sequenced and compared to the homologous genes from Caenorhabditis elegans. The direct tandem organization of these genes, gpd-2 and gpd-3, and the size and location of the two introns in each gene are the same in C. elegans and C. briggsae. Primer-extension studies demonstrated that the two genes in C. briggsae are trans-splice differentially with the same splice leader (SL) RNAs as are observed in C. elegans. The gdp-2 gene is trans-spliced with SL1 while gdp-3 is trans-spliced with SL2. Significant sequence conservation was observed within the promoter regions of each species and may indicate those regions responsible for body-wall-muscle-specific gene expression and/or differential trans-splicing. Comparisons of the sequences suggest that the tandem repeat of the genes has been subjected to concerted evolution and that C. briggsae and C. elegans diverged much earlier than would be anticipated based on morphological similarities alone. Finally, an open reading frame found several hundred nucleotides upstream from gpd-2, in both species, appears to be homologous to the ATP synthase subunit, ATPase inhibitor protein, from bovine mitochondria.