Neurotoxin localization to ectodermal gland cells uncovers an alternative mechanism of venom delivery in sea anemones

Jellyfish, hydras, corals and sea anemones (phylum Cnidaria) are known for their venomous stinging cells, nematocytes, used for prey and defence. Here we show, however, that the potent Type I neurotoxin of the sea anemone Nematostella vectensis, Nv1, is confined to ectodermal gland cells rather than nematocytes. We demonstrate massive Nv1 secretion upon encounter with a crustacean prey. Concomitant discharge of nematocysts probably pierces the prey, expediting toxin penetration. Toxin efficiency in sea water is further demonstrated by the rapid paralysis of fish or crustacean larvae upon application of recombinant Nv1 into their medium. Analysis of other anemone species reveals that in Anthopleura elegantissima, Type I neurotoxins also appear in gland cells, whereas in the common species Anemonia viridis, Type I toxins are localized to both nematocytes and ectodermal gland cells. The nematocyte-based and gland cell-based envenomation mechanisms may reflect substantial differences in the ecology and feeding habits of sea anemone species. Overall, the immunolocalization of neurotoxins to gland cells changes the common view in the literature that sea anemone neurotoxins are produced and delivered only by stinging nematocytes, and raises the possibility that this toxin-secretion mechanism is an ancestral evolutionary state of the venom delivery machinery in sea anemones.     

Cnidarians and ancestral genetic complexity in the animal kingdom

Eleven of the twelve recognized wingless (Wnt) subfamilies are represented in the sea anemone Nematostella vectensis, indicating that this developmentally important gene family was already fully diversified in the common ancestor of 'higher' animals. In deuterostomes, although duplications have occurred, no novel subfamilies of Wnts have evolved. By contrast, the protostomes Drosophila and Caenorhabditis have lost half of the ancestral Wnts. This pattern -- loss of genes from an ancestrally complex state -- might be more important in animal evolution than previously recognized.     

Concerted evolution of two novel protein families in Caenorhabditis species

Among a large number of homologous gene clusters in C. elegans, two gene families that appear to undergo concerted evolution were studied in detail. Both gene families are nematode specific and encode small secreted proteins of unknown function. For both families in three Caenorhabditis species, concerted groups of genes are characterized by close genomic proximity and by genes in inverted orientation. The rate of protein evolution in one of the two families could be calibrated by comparison with a closely related nonconcerted singleton gene with one-to-one orthologs in all three species. This comparison suggests that protein evolution in concerted gene clusters is two- to sevenfold accelerated. A broader survey of clustered gene families, focused on adjacent inverted gene pairs, identified an additional seven families in which concerted evolution probably occurs. All nine identified families encode relatively small proteins, eight of them encode putative secreted proteins, and most of these have very unusual amino acid composition or sequence. I speculate that these genes encode rapidly evolving antimicrobial peptides.     

When positive selection of neurotoxin genes is missing. The riddle of the sea anemone Nematostella vectensis

Rapid evolution driven by positive Darwinian selection appears in toxins of vipers, scorpions, and marine snails. Although the vast phylogenetic distances between these animals suggest that this phenomenon is common, the recent release of the genome of Nematostella vectensis (Starlet anemone) as a collection of contigs portrays another extreme. Besides potassium channel toxin domains, which resemble potassium channel blockers, embedded in various genes, only one gene family encoding for sodium channel neurotoxins has been found, and the putative mature product of 10 family members is identical. Whereas the existence of a single toxin encoded by multiple genes may be explained by the unique ecology of N. vectensis, the complete absence of substitutions including synonymous ones is surprising and suggests either that these genes have been duplicated recently, or that their total conservation was advantageous. A retro-element identified downstream to one of the genes offers a possible mechanism of enhanced toxin gene duplication. This assumption still awaits further verification as soon as the various contigs are assigned within larger genomic fragments.     

Coevolution of immunoglobulin heavy- and light-chain variable-region gene families

The gene families encoding the immunoglobulin variable regions of heavy (VH) and light (VL) chains in vertebrates are composed of many genes. However, the gene number and the extent of diversity among VH and VL gene copies vary with species. To examine the causes of this variation and the evolutionary forces for these multigene families, we conducted a phylogenetic analysis of VH and VL genes from the species of amniotes. The results of our analysis showed that for each species, VH and VL genes have the same pattern of clustering in the trees, and, according to this clustering pattern, the species can be divided into two groups. In the first group of species (humans and mice), VH and VL genes were extensively intermingled with genes from other organisms; in the second group of species (chickens, rabbits, cattle, sheep, swine, and horses), the genes tended to form clusters within the same group of organisms. These results suggest that the VH and VL multigene families have evolved in the same fashion: they have undergone coordinated contraction and expansion of gene repertoires such that each group of organisms is characterized by a certain level of diversity of VH and VL genes. The extent of diversity among copies of VH and VL genes in each species is related to the mechanism of generation of antibody variety. In humans and mice, DNA rearrangement of immunoglobulin variable, diversity, and joining-segment genes is a main source of antibody diversity, whereas in chickens, rabbits, cattle, sheep, swine, and horses, somatic hypermutation and somatic gene conversion play important roles. The evolutionary pattern of VH and VL multigene families is consistent with the birth-and-death model of evolution, yet different levels of diversifying selection seem to operate in the VH and VL genes of these two groups of species.      

Patterns of divergence of a large family of nodule cysteine‐rich peptides in accessions of Medicago truncatula

The nodule cysteine‐rich (NCR) groups of defensin‐like (DEFL) genes are one of the largest gene families expressed in the nodules of some legume plants. They have only been observed in the inverted repeat loss clade (IRLC) of legumes, which includes the model legume Medicago truncatula. NCRs are reported to play an important role in plant–microbe interactions. To understand their diversity we analyzed their expression and sequence polymorphisms among four accessions of M. truncatula. A significant expression and nucleotide variation was observed among the genes. We then used 26 accessions to estimate the selection pressures shaping evolution among the accessions by calculating the nucleotide diversity at non‐synonymous and synonymous sites in the coding region. The mature peptides of the orthologous NCRs had signatures of both purifying and diversifying selection pressures, unlike the seed DEFLs, which predominantly exhibited purifying selection. The expression, sequence variation and apparent diversifying selection in NCRs within the Medicago species indicates rapid and recent evolution, and suggests that this family of genes is actively evolving to adapt to different environments and is acquiring new functions.     

Diverse evolutionary mechanisms shape the type III effector virulence factor repertoire in the plant pathogen Pseudomonas syringae

Many gram-negative pathogenic bacteria directly translocate effector proteins into eukaryotic host cells via type III delivery systems. Type III effector proteins are determinants of virulence on susceptible plant hosts; they are also the proteins that trigger specific disease resistance in resistant plant hosts. Evolution of type III effectors is dominated by competing forces: the likely requirement for conservation of virulence function, the avoidance of host defenses, and possible adaptation to new hosts. To understand the evolutionary history of type III effectors in Pseudomonas syringae, we searched for homologs to 44 known or candidate P. syringae type III effectors and two effector chaperones. We examined 24 gene families for distribution among bacterial species, amino acid sequence diversity, and features indicative of horizontal transfer. We assessed the role of diversifying and purifying selection in the evolution of these gene families. While some P. syringae type III effectors were acquired recently, others have evolved predominantly by descent. The majority of codons in most of these genes were subjected to purifying selection, suggesting selective pressure to maintain presumed virulence function. However, members of 7 families had domains subject to diversifying selection.     

Genetic diversity of MHC class I loci in six non-model frogs is shaped by positive selection and gene duplication

Comparative studies of major histocompatibility complex (MHC) genes across vertebrate species can reveal the evolutionary processes that shape the structure and function of immune regulatory proteins. In this study, we characterized MHC class I sequences from six frog species representing three anuran families (Hylidae, Centrolenidae and Ranidae). Using cDNA from our focal species, we amplified a total of 79 unique sequences spanning exons 2-4 that encode the extracellular domains of the functional alpha chain protein. We compared intra- and interspecific nucleotide and amino-acid divergence, tested for recombination, and identified codon sites under selection by estimating the rate of non-synonymous to synonymous substitutions with multiple codon-based maximum likelihood methods. We determined that positive (diversifying) selection was acting on specific amino-acid sites located within the domains that bind pathogen-derived peptides. We also found significant signals of recombination across the physical distance of the genes. Finally, we determined that all the six species expressed two or three putative classical class I loci, in contrast to the single locus condition of Xenopus laevis. Our results suggest that MHC evolution in anurans is a dynamic process and that variation in numbers of loci and genetic diversity can exist among taxa. Thus, the accumulation of genetic data for more species will be useful in further characterizing the relative importance of processes such as selection, recombination and gene duplication in shaping MHC loci among amphibian lineages.     

Evolving disease resistance genes

Defenses against most specialized plant pathogens are often initiated by a plant disease resistance gene. Plant genomes encode several classes of genes that can function as resistance genes. Many of the mechanisms that drive the molecular evolution of these genes are now becoming clear. The processes that contribute to the diversity of R genes include tandem and segmental gene duplications, recombination, unequal crossing-over, point mutations, and diversifying selection. Diversity within populations is maintained by balancing selection. Analyses of whole-genome sequences have and will continue to provide new insight into the dynamics of resistance gene evolution.     

Evolutionary Analyses Suggest a Function of MxB Immunity Proteins Beyond Lentivirus Restriction

Viruses impose diverse and dynamic challenges on host defenses. Diversifying selection of codons and gene copy number variation are two hallmarks of genetic innovation in antiviral genes engaged in host-virus genetic conflicts. The myxovirus resistance (Mx) genes encode interferon-inducible GTPases that constitute a major arm of the cell-autonomous defense against viral infection. Unlike the broad antiviral activity of MxA, primate MxB was recently shown to specifically inhibit lentiviruses including HIV-1. We carried out detailed evolutionary analyses to investigate whether genetic conflict with lentiviruses has shaped MxB evolution in primates. We found strong evidence for diversifying selection in the MxB N-terminal tail, which contains molecular determinants of MxB anti-lentivirus specificity. However, we found no overlap between previously-mapped residues that dictate lentiviral restriction and those that have evolved under diversifying selection. Instead, our findings are consistent with MxB having a long-standing and important role in the interferon response to viral infection against a broader range of pathogens than is currently appreciated. Despite its critical role in host innate immunity, we also uncovered multiple functional losses of MxB during mammalian evolution, either by pseudogenization or by gene conversion from MxA genes. Thus, although the majority of mammalian genomes encode two Mx genes, this apparent stasis masks the dramatic effects that recombination and diversifying selection have played in shaping the evolutionary history of Mx genes. Discrepancies between our study and previous publications highlight the need to account for recombination in analyses of positive selection, as well as the importance of using sequence datasets with appropriate depth of divergence. Our study also illustrates that evolutionary analyses of antiviral gene families are critical towards understanding molecular principles that govern host-virus interactions and species-specific susceptibility to viral infection.     

Diversifying selection of the tumor-growth promoter angiogenin in primate evolution

Diversifying selection drives the rapid differentiation of gene sequences and is one of the main forces behind adaptive evolution. Most genes known to be shaped by diversifying selection are those involved in host-pathogen or male-female interactions characterized as molecular "arms races." Here we report the unexpected detection of diversifying selection in the evolution of a tumor-growth promoter, angiogenin (ANG). A comparison among 11 primate species demonstrates that ANG has a significantly higher rate of nucleotide substitution at nonsynonymous sites than at synonymous sites, a hallmark of positive selection acting at the molecular level. Furthermore, we observed significant charge diversity at the molecular surface, suggesting the presence of selective pressures in the microenvironment of ANG, including its binding molecules. A population survey of ANG in chimpanzees, however, reveals no polymorphism, which may have resulted from a recent selective sweep of a charge-altering substitution in chimpanzee evolution. Functional assays of recombinant ANGs from the human and owl monkey indicate that primate ANGs retain angiogenic activity despite rapid evolution. Our study, together with findings of similar selection in the primate breast cancer suppressor gene, BRCA1, reveals an intriguing phenomenon of unusual selective pressures on, and adaptive evolution of, cancer-related genes in primate evolution.     

Identification and characterisation of a novel immune-type receptor (NITR) gene cluster in the European sea bass, Dicentrarchus labrax, reveals recurrent gene expansion and diversification by positive selection

In the last decade, a new gene family encoding non-rearranging receptors, called novel immune-type receptors (NITRs), has been discovered in teleost fish. NITRs belong to the immunoglobulin superfamily and represent an extraordinarily divergent and rapidly evolving gene complex. Genomic analysis of a region spanning 270 kb led to the discovery of a NITR gene cluster in the European sea bass (Dicentrarchus labrax). In total, 27 NITR genes and three putative pseudogenes, organised in a tandemly arrayed cluster, were identified. Sea bass NITR genes maintain the three major genomic organisations that appear to be essentially conserved among fish species along with new features presumably involving processes of intron loss, exon deletion and acquisition of new exons. Comparative and evolutionary analyses suggest that these receptors have evolved following a “birth-and-death” model of gene evolution in which duplication events together with lineage-specific gain and loss of individual members contributed to the rapid diversification of individual gene families. In this study, we demonstrate that species-specific gene expansions provide the raw material for diversifying, positive Darwinian selection favouring the evolution of a highly diverse array of molecules.     

Gene family evolution across 12 Drosophila genomes

Comparison of whole genomes has revealed large and frequent changes in the size of gene families. These changes occur because of high rates of both gene gain (via duplication) and loss (via deletion or pseudogenization), as well as the evolution of entirely new genes. Here we use the genomes of 12 fully sequenced Drosophila species to study the gain and loss of genes at unprecedented resolution. We find large numbers of both gains and losses, with over 40% of all gene families differing in size among the Drosophila. Approximately 17 genes are estimated to be duplicated and fixed in a genome every million years, a rate on par with that previously found in both yeast and mammals. We find many instances of extreme expansions or contractions in the size of gene families, including the expansion of several sex- and spermatogenesis-related families in D. melanogaster that also evolve under positive selection at the nucleotide level. Newly evolved gene families in our dataset are associated with a class of testes-expressed genes known to have evolved de novo in a number of cases. Gene family comparisons also allow us to identify a number of annotated D. melanogaster genes that are unlikely to encode functional proteins, as well as to identify dozens of previously unannotated D. melanogaster genes with conserved homologs in the other Drosophila. Taken together, our results demonstrate that the apparent stasis in total gene number among species has masked rapid turnover in individual gene gain and loss. It is likely that this genomic revolving door has played a large role in shaping the morphological, physiological, and metabolic differences among species.     

vasa and nanos expression patterns in a sea anemone and the evolution of bilaterian germ cell specification mechanisms

Most bilaterians specify primordial germ cells (PGCs) during early embryogenesis using either inherited cytoplasmic germ line determinants (preformation) or induction of germ cell fate through signaling pathways (epigenesis). However, data from nonbilaterian animals suggest that ancestral metazoans may have specified germ cells very differently from most extant bilaterians. Cnidarians and sponges have been reported to generate germ cells continuously throughout reproductive life, but previous studies on members of these basal phyla have not examined embryonic germ cell origin. To try to define the embryonic origin of PGCs in the sea anemone Nematostella vectensis, we examined the expression of members of the vasa and nanos gene families, which are critical genes in bilaterian germ cell specification and development. We found that vasa and nanos family genes are expressed not only in presumptive PGCs late in embryonic development, but also in multiple somatic cell types during early embryogenesis. These results suggest one way in which preformation in germ cell development might have evolved from the ancestral epigenetic mechanism that was probably used by a metazoan ancestor.     

Morphological and molecular analysis of the Nematostella vectensis cnidom

The starlet sea anemone Nematostella vectensis is an emerging model organism for developmental and evolutionary biology. Due to the availability of genome data and its amenability to genetic manipulation Nematostella serves as a source for comparative molecular and phylogenetic studies. Despite this fact, the characterization of the nematocyst inventory and of nematocyst-specific genes is still fragmentary and sometimes misleading in this cnidarian species. Here, we present a thorough qualitative and quantitative analysis of nematocysts in Nematostella vectensis. In addition, we have cloned major nematocyst components, Nematostella minicollagens 1, 3 and 4, and show their expression patterns by in situ hybridization and immunocytochemistry using specific antibodies. Our data provides tools and insights for further studies on nematocyst morphogenesis in Nematostella and comparative evolution in cnidarians.     

Receptor-like genes in the major resistance locus of lettuce are subject to divergent selection.

Disease resistance genes in plants are often found in complex multigene families. The largest known cluster of disease resistance specificities in lettuce contains the RGC2 family of genes. We compared the sequences of nine full-length genomic copies of RGC2 representing the diversity in the cluster to determine the structure of genes within this family and to examine the evolution of its members. The transcribed regions range from at least 7.0 to 13.1 kb, and the cDNAs contain deduced open reading frames of approximately 5. 5 kb. The predicted RGC2 proteins contain a nucleotide binding site and irregular leucine-rich repeats (LRRs) that are characteristic of resistance genes cloned from other species. Unique features of the RGC2 gene products include a bipartite LRR region with >40 repeats. At least eight members of this family are transcribed. The level of sequence diversity between family members varied in different regions of the gene. The ratio of nonsynonymous (Ka) to synonymous (Ks) nucleotide substitutions was lowest in the region encoding the nucleotide binding site, which is the presumed effector domain of the protein. The LRR-encoding region showed an alternating pattern of conservation and hypervariability. This alternating pattern of variation was also found in all comparisons within families of resistance genes cloned from other species. The Ka /Ks ratios indicate that diversifying selection has resulted in increased variation at these codons. The patterns of variation support the predicted structure of LRR regions with solvent-exposed hypervariable residues that are potentially involved in binding pathogen-derived ligands.     

Cryptic complexity captured: the Nematostella genome reveals its secrets

The full genomic sequence of the sea anemone Nematostella vectensis, which is the first full genomic sequence for a representative of the Phylum Cnidaria, has recently been published, providing some surprising findings and a unique perspective on the evolution of animal genomes. Major conclusions are that, in gene number, composition and intron/exon structure, the anemone is more similar to vertebrates than are flies and nematodes and that this shared complexity must therefore be very ancient.