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.     

Molecular evolution and quantitative variation for chemosensory behaviour in the nematode genus Caenorhabditis

Caenorhabditis elegans is a model organism in biology, yet despite the tremendous information generated from genetic, genomic and functional analyses, C. elegans has rarely been used to address questions in ecological genetics. Here, we analyse genetic variation for chemosensory behaviour, an ecologically important trait that is also genetically well characterized, at both the phenotypic and molecular levels within three species of the genus Caenorhabditis. We show that the G-protein ODR-3 plays an important role in chemosensory avoidance behaviour and identify orthologues of odr-3 in C. briggsae and C. remanei. Both quantitative genetic analysis of chemosensory behaviour and molecular population genetic analysis of odr-3 show that there is little genetic variation among a worldwide collection of isolates of the primarily selfing C. elegans, whereas there is substantially more variation within a single population of the outcrossing C. remanei. Although there are a large number of substitutions at silent sites within odr-3 among the three species, molecular evolution at the protein level is extremely conserved, suggesting that odr-3 plays an important role in cell signalling during chemosensation and/or neuronal cilia development in C. remanei and in C. briggsae as it does in C. elegans. Our results suggest that C. remanei may be a more suitable subject for ecological and evolutionary genetic studies than C. elegans.     

Evolution of the vertebrate ABC gene family: analysis of gene birth and death

Vertebrate evolution has been largely driven by the duplication of genes that allow for the acquisition of new functions. The ATP-binding cassette (ABC) proteins constitute a large and functionally diverse family of membrane transporters. The members of this multigene family are found in all cellular organisms, most often engaged in the translocation of a wide variety of substrates across lipid membranes. Because of the diverse function of these genes, their large size, and the large number of orthologs, ABC genes represent an excellent tool to study gene family evolution. We have identified ABC proteins from the sea squirt (Ciona intestinalis), zebrafish (Danio rerio), and chicken (Gallus gallus) and, using phylogenetic analysis, identified those genes with a one-to-one orthologous relationship to human ABC proteins. All ABC protein subfamilies found in Ciona and zebrafish correspond to the human subfamilies, with the exception of a single ABCH subfamily gene found only in zebrafish. Multiple gene duplication and deletion events were identified in different lineages, indicating an ongoing process of gene evolution. As many ABC genes are involved in human genetic diseases, and important drug transport phenotypes, the understanding of ABC gene evolution is important to the development of animal models and functional studies.     

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.     

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.     

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.     

Evidence for active maintenance of inverted repeat structures identified by a comparative genomic approach

Inverted repeats have been found to occur in both prokaryotic and eukaryotic genomes. Usually they are short and some have important functions in various biological processes. However, long inverted repeats are rare and can cause genome instability. Analyses of C. elegans genome identified long, nearly-perfect inverted repeat sequences involving both divergently and convergently oriented homologous gene pairs and complete intergenic sequences. Comparisons with the orthologous regions from the genomes of C. briggsae and C. remanei show that the inverted repeat structures are often far more conserved than the sequences. This observation implies that there is an active mechanism for maintaining the inverted repeat nature of the sequences.     

Rapid coevolution of the nematode sex-determining genes fem-3 and tra-2

Unlike many features of metazoan development, sex determination is not widely conserved among phyla. However, the recent demonstration that one gene family controls sexual development in Drosophila, C. elegans, and vertebrates suggests that sex determination mechanisms may have evolved from a common pathway that has diverged radically since the Cambrian. Sex determination gene sequences often evolve quickly, but it is not known how this relates to higher-order pathways or what selective or neutral forces are driving it. In such a rapidly evolving developmental pathway, the fate of functionally linked genes is of particular interest. To investigate a pair of such genes, we cloned orthologs of the key C. elegans male-promoting gene fem-3 from two sister species, C. briggsae and C. remanei. We employed RNA interference to show that in all three species, the male-promoting function of fem-3 and its epistatic relationship with its female-promoting upstream repressor, tra-2, are conserved. Consistent with this, the FEM-3 protein interacts with TRA-2 in each species, but in a strictly species-specific manner. Because FEM-3 is the most divergent protein yet described in Caenorhabditis and the FEM-3 binding domain of TRA-2 is itself hypervariable, a key protein-protein interaction is rapidly evolving in concert. Extrapolation of this result to larger phylogenetic scales helps explain the dissimilarity of the sex determination systems across phyla.     

Mode and Tempo of Molecular Evolution in the Nematode Caenorhabditis: Cytochrome Oxidase II and Calmodulin Sequences

Through direct sequencing methods, the mitochondrial gene for cytochrome oxidase subunit two (CO II) and the single-copy nuclear gene for calmodulin were compared among strains of Caenorhabidits elegans and two other Caenorhabditis species (C. remanei and C. briggsae). In addition the CO II sequence was determined from a distantly related nematode, Steinernema intermedii. Among the 11 strains of C. elegans tested, there are four types of CO II gene, arising from two major lineages. Levels of intraspecific difference in the CO II gene are low (less than 2.0%) compared to the extraordinary divergence between congeneric species, which is about 50% when corrected for multiple hits. Concordant with the increase in divergence between taxa is a change in the pattern of substitution from a strong transition bias (24 transitions compared to two transversions) within species to a substitution pattern that appears to reflect the base composition of the mitochondrial genome when more divergent nematodes are compared. The base composition of the Caenorhabditis CO II gene is strongly biased toward A + T at all three positions of codons and appears to constrain the amino acid composition of the protein. Both the CO II and calmodulin genes show extreme conservation of amino acid sequences. When the accumulation of changes at silent sites in the two genes is compared among strains, it becomes evident that the mitochondrial gene is changing faster than the nuclear gene.     

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.     

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.     

A family of zinc-finger proteins is required for chromosome-specific pairing and synapsis during meiosis in C. elegans

Homologous chromosome pairing and synapsis are prerequisite for accurate chromosome segregation during meiosis. Here, we show that a family of four related C2H2 zinc-finger proteins plays a central role in these events in C. elegans. These proteins are encoded within a tandem gene cluster. In addition to the X-specific HIM-8 protein, three additional paralogs collectively mediate the behavior of the five autosomes. Each chromosome relies on a specific member of the family to pair and synapse with its homolog. These "ZIM" proteins concentrate at special regions called meiotic pairing centers on the corresponding chromosomes. These sites are dispersed along the nuclear envelope during early meiotic prophase, suggesting a role analogous to the telomere-mediated meiotic bouquet in other organisms. To gain insight into the evolution of these components, we characterized homologs in C. briggsae and C. remanei, which revealed changes in copy number of this gene family within the nematode lineage.     

Cloning by synteny: identifying C. briggsae homologues of C. elegans genes.

Phylogenetic comparisons of gene and protein sequences between related species are often used to identify evolutionarily conserved elements that are important for gene expression, function, or regulation. However, homologoues may sometimes be difficult to identify by conventional low stringency hybridisation techniques, if they have undergone substantial sequence divergence. A new approach, cloning by synteny, is described that was used to identify the C. briggsae homologue of the C. elegans sex-determining gene tra-2. We show that four genes tra-2, ppp-1, art-1, and sod-1 are organised in a syntenic cluster and suggest that extensive conservation of gene linkage may exist between C. briggsae and C. elegans. We have also constructed a C. briggsae cDNA library to facilitate characterisation of these genes. Given the rapid progress in the physical mapping and sequencing of the C. elegans genome, cloning by synteny may provide the fastest method for identifying C. briggsae gene homologues, especially for genes encoding novel proteins.     

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.     

Smoke without fire: most reported cases of intron gain in nematodes instead reflect intron losses

Identification of recently gained spliceosomal introns would provide crucial evidence in the continuing debate concerning the age and evolutionary significance of introns. A previously published genomic analysis reported to have identified 122 introns that had been gained since the divergence of the nematodes Caenorhabidits elegans and Caenorhabditis briggsae approximately 100 MYA. However, using newly available genomic sequence from additional Caenorhabditis species, we show that 74% (60/81) of the reported gains in C. elegans are present in a C. briggsae relative. This pattern indicates that these introns represent losses in C. briggsae, not gains in C. elegans. In addition, 61% (25/41) of the reported gains in C. briggsae are present in the more distant C. briggsae relative, in a pattern suggesting that additional reported gains in C. elegans and/or C. briggsae may in fact represent unrecognized losses. These results underscore the dominance of intron loss over intron gain in recent eukaryotic evolution, the pitfalls associated with parsimony in inferring intron gains, and the importance of genomic sequencing of clusters of closely related species for drawing accurate inferences about genome evolution.     

Clustered organization of reproductive genes in the C. elegans genome

Defining the forces that sculpt genome organization is fundamental for understanding the origin, persistence, and diversification of species. The genomic sequences of the nematodes Caenorhabditis elegans and Caenorhabditis briggsae provide an excellent opportunity to explore the dynamics of chromosome evolution. Extensive chromosomal rearrangement has accompanied divergence from their common ancestor, an event occurring roughly 100 million years ago (Mya); yet, morphologically, these species are nearly indistinguishable and both reproduce primarily by self-fertilization. Here, we show that genes expressed during spermatogenesis (sperm genes) are nonrandomly distributed across the C. elegans genome into three large clusters located on two autosomes. In addition to sperm genes, these chromosomal regions are enriched for genes involved in the hermaphrodite sperm/oocyte switch and in the reception of sperm signals that control fertilization. Most loci are present in single copy, suggesting that cluster formation is largely due to gene aggregation and not to tandem duplication. Comparative mapping indicates that the C. briggsae genome differs dramatically from the C. elegans genome in clustering. Because clustered genes have a direct role in reproduction and thus fitness, their aggregated pattern might have been shaped by natural selection, perhaps as hermaphroditism evolved.