Concerted Evolution of Sea Anemone Neurotoxin Genes Is Revealed through Analysis of the Nematostella vectensis Genome

Abstract

Gene families, which encode toxins, are found in many poisonous animals, yet there is limited understanding of their evolution at the nucleotide level. The release of the genome draft sequence for the sea anemone Nematostella vectensis enabled a comprehensive study of a gene family whose neurotoxin products affect voltage-gated sodium channels. All gene family members are clustered in a highly repetitive ∼30-kb genomic region and encode a single toxin, Nv1. These genes exhibit extreme conservation at the nucleotide level which cannot be explained by purifying selection. This conservation greatly differs from the toxin gene families of other animals (e.g., snakes, scorpions, and cone snails), whose evolution was driven by diversifying selection, thereby generating a high degree of genetic diversity. The low nucleotide diversity at the Nv1 genes is reminiscent of that reported for DNA encoding ribosomal RNA (rDNA) and 2 hsp70 genes from Drosophila, which have evolved via concerted evolution. This evolutionary pattern was experimentally demonstrated in yeast rDNA and was shown to involve unequal crossing-over. Through sequence analysis of toxin genes from multiple N. vectensis populations and 2 other anemone species, Anemonia viridis and Actinia equina, we observed that the toxin genes for each sea anemone species are more similar to one another than to those of other species, suggesting they evolved by manner of concerted evolution. Furthermore, in 2 of the species (A. viridis and A. equina) we found genes that evolved under diversifying selection, suggesting that concerted evolution and accelerated evolution may occur simultaneously.

Introduction

Large gene families encoding for diverse peptide toxins are common in poisonous animals such as snakes, cone snails, scorpions, and sea anemones (Duda and Palumbi 1999; Froy et al. 1999; Honma and Shiomi 2006; Lynch 2007). In the first 3 groups of animals, some of the gene family members have evolved in an accelerated manner when compared with other regions of the genome, presumably due to positive Darwinian selection (also known as diversifying selection; Nakashima et al. 1993; Duda and Palumbi 1999; Zhu et al. 2004). Nevertheless, the study of toxin gene families and their evolution is still limited, mostly because nucleotide data, especially at the level of genomic DNA (gDNA), are scarce. Sea anemones (Anthozoa: Actinaria) are ancient predators (Chen et al. 2002) which live in marine habitats worldwide. They are members of the Cnidaria, which diverged from the lineage leading to the Bilateria at a very early stage in evolution, over 600 MYA (Ball et al. 2004). The ancient divergence as well as the study of various developmental stages in the starlet anemone, Nematostella vectensis, prompted the sequencing of its entire genome (Darling et al. 2005; Sullivan et al. 2006; Putnam et al. 2007). This has provided a unique opportunity to analyze the evolution of a neurotoxin gene family. Sea anemones produce a variety of highly potent modulators of voltage-dependent sodium channels including Type I and Type II neurotoxins capable of paralyzing and killing prey or foe (Honma and Shiomi 2006). By conducting a Blast homology search of these toxin sequences on the previous release of N. vectensis genome draft in the form of annotated contigs (www.stellabase.org; Sullivan et al. 2006), we discovered 11 genes encoding for a single putative neurotoxin (named Nv1; Moran and Gurevitz 2006). The toxic potential of Nv1 was verified by expressing it in recombinant form followed by functional analysis (Moran Y and Gurevitz M, unpublished data). The structure of Nv1 genes resembles previously studied homologous genes of the sea anemone Calliactis parasitica, clx-1 and clx-2 (Spagnuolo et al. 1994). The genes encoding these toxins include 2 exons separated by an intron. The first exon encodes for a leader peptide and a propart (a peptide region cleaved upon toxin maturation), and the second encodes for the end of the propart and the mature toxin (Spagnuolo et al. 1994; Anderluh et al. 2000). Although some of these 11 genes differ slightly in sequence (0–2.5% variability) of the intron and first exon, the region encoding for the mature toxin is identical. This identity is surprising when compared with the highly variable multiple toxin genes of other poisonous animals (Nakashima et al. 1993; Duda and Palumbi 1999; Zhu et al. 2004).

Unlike the tight conservation of protein products due to purifying selection observed in many other gene families (Nei and Rooney 2005), the unusual conservation at the nucleotide level of the Nv1 genes is reminiscent of genes encoding for ribosomal RNA (rDNA). The rDNA copies and their surrounding regions in a single species are all more similar to one another than to rDNA genes of other species (Nei and Rooney 2005; Eickbush TH and Eickbush DG 2007; Ganley and Kobayashi 2007). This phenomenon, first reported in rDNA of Xenopus species (Brown et al. 1972), was explained by a mechanism termed “concerted evolution,” in which a novel mutation in 1 gene copy is either passed to all other gene copies or reversed back by the unmutated copies (Nei and Rooney 2005; Eickbush TH and Eickbush DG 2007). Concerted evolution was experimentally shown in the yeast Saccharomyces cerevisiae (Szostak and Wu 1980) to be based on unequal crossover. In addition, gene conversion is also considered a possible mechanism involved in concerted evolution, but no experimental proof is available. A number of proposed examples of gene families evolving through concerted evolution have not been supported with further analysis (Nei and Rooney 2005), leaving rDNA and a tandem of heat shock proteins from Drosophila melanogaster as the only examples currently supported.

The release of the N. vectensis genome in larger resolved scaffolds (Putnam et al. 2007; http://genome.jgi-psf.org/Nemve1/Nemve1.home.html) enabled us to revisit the genomic landscape that contains the Nv1 genes in order to examine their evolutionary pattern. Moreover, to evaluate the extent of nucleotide conservation of nv1 (the genes encoding the Nv1 toxin), we analyzed neurotoxin-encoding genes from 2 other N. vectensis populations, both genetically and geographically distinct from the sequenced population from Rhode River, MD (Darling et al. 2004; Reitzel et al. 2007). Because N. vectensis dwells in a specialized habitat (brackish lagoons and estuaries), we also isolated and analyzed DNA and mRNA sequences of toxin genes from 2 Mediterranean sea anemones, Anemonia viridis (snakelocks anemone) and Actinia equina (beadlet anemone). Av2 (previously known as ATX-II) is a highly potent toxin of A. viridis and is widely used in neurological research (Honma and Shiomi 2006). Although it was the first type I sea anemone toxin to be amino acid sequenced (Wunderer et al. 1976), no data are available on the gene encoding it as sea anemone toxin genes and transcripts were rarely studied. One of the few exceptions is the mRNA encoding for the Ae1 toxin isolated from the tentacles of A. equina (Lin et al. 1996; Anderluh et al. 2000), which was instrumental for gene analysis.

Analysis and comparison of the N. vectensis genomic data and the data from other N. vectensis populations and other anemone species indicated much higher similarity among toxin genes within each species than the similarity of these genes when compared between 2 species. This pattern of intraspecies conservation implies that sea anemone type I toxin genes evolved in a concerted manner. Moreover, we provide evidence that positive Darwinian selection conditions may have also affected the evolution of these toxin genes.

Materials and Methods

Biological Strains and Samples

All DNA manipulations and plasmid preparations were performed using the Escherichia coli strain DH5α. Actinia equina from Rosh-Hanikra, Israel and Anemonia viridis from Atlit beach, Israel were kind gifts from G. Mor and N. Lazarus (Tel Aviv University), respectively. They were kept alive in seawater, swiftly dried in paper towels, and frozen at −70 °C until used. Nematostella vectensis adults were collected from tidally restricted pools at Crane Beach and Neponset River Estuary (Massachusetts, USA) and placed in sterile culture conditions (13 parts per thousand, Instant Ocean artificial seawater). Individuals were fed freshly hatched Artemia 2–3 times per week.

Extraction of Nucleic Acids

Actinia equina and Anemonia viridis were flash frozen in liquid nitrogen and ground into a fine powder using mortar and pestle. For DNA extraction, the powder was dissolved in extraction buffer (Tris 10 mM, pH 8.0, ethylenediaminetetraacetic acid 100 mM, sodium dodecyl sulfate 0.5%) with proteinase K 0.5 mg/ml (Sigma, St Louis, MO) and then incubated at 65 °C for 2 and a half hours before the addition of 10% Cetrimonium (hexadecyltrimethylammonium) bromide (Sigma) in 0.7 M NaCl to a final concentration of 0.3% of the sample volume. The samples were then incubated for 20 additional minutes at the same conditions and then an equal volume of chloroform/isoamyl alcohol (24:1) was added, mixed, and centrifuged at 12,000 × g for 20 min. From this point forward, the procedure was continued as previously described (Dellacorte 1994). For RNA extraction, the powder was dissolved in Trizol reagent (Invitrogen, Carlsbad, CA), and further RNA purification steps were carried according to the manufacturer's instructions. The RNA was treated with a “DNA Free kit” (Ambion, Austin, TX) in order to eliminate any residual DNA. Each aliquot of nucleic acids from A. equina or A. viridis was produced from a single individual. Nematostella vectensis adults were starved for at least 7 days prior to extraction of DNA using the DNeasy blood and tissue kit (Qiagen, Valencia, CA).

3′ and 5′ Rapid Amplification of cDNA Ends

Both 3′ and 5′ rapid amplification of cDNA ends (RACE) were performed by the 5′/3′ RACE kit, second generation (Roche Applied Sciences, Mannheim, Germany) according to the manufacturer's instructions in the presence of Protector RNAse inhibitor (Roche). The single-stranded DNA obtained was amplified by polymerase chain reaction (PCR) using the Pwo enzyme (Roche), and the products were cloned into pBluescript KS (Stratagene, La Jolla, CA) predigested with EcoRV. Degenerate primers were used for amplifying the sequence encoding the mature Av2, which was later verified by PCR using nondegenerate primers corresponding to the transcript ends.

Genome Walking

Genome walking was performed as previously described (Siebert et al. 1995). Briefly, 2.5 μg of A. viridis gDNA was digested with DraI, EcoRV, ScaI, or StuI (New England Biolabs, Ipswich, MA), cleaned, and ligated by Mighty mix ligation kit (Takara Bio, Shiga, Japan) to DNA adapters. Touchdown PCR was performed with a primer corresponding to the adapter and a primer corresponding to the sequence of the gene of interest using the digested gDNA as template. This stage was followed by nested PCR, using the diluted product of the first PCR as a template. All genome walking PCRs were performed with Phusion, hot start version (Finnzymes, Espoo, Finland), or LA taq (Takara Bio), and the products were cloned to pBluescript KS (Stratagene) digested with EcoRV or pGEM-T (Promega, Madison, Wisconsin), respectively.

Local Alignment Search

All local alignment searches were performed using BlastN, BlastX, or BlastP v. 2.2.17 (Altschul et al. 1997) based on BLOSUM62 matrix. Searches in the N. vectensis genome draft were performed via the Joint Genome Institute Web site (Putnam et al. 2007; http://genome.jgi-psf.org/Nemve1/Nemve1.home.html), from which the gene and scaffold nomenclature of N. vectensis are taken.

Multiple Alignments, Tests of Selection, and Phylogeny

Multiple alignments were created using CLC Free Workbench 4.0 (CLC bio, Aarhus, Denmark) with the following parameters: gap open cost = 10, gap extension cost = 1, end gap cost = as any other. Alignments were converted to ClustalW format and analyzed by MEGA 3.1 (Kumar et al. 2004). Inference for selection (dN/dS ratios) was based on 3 large Z-tests of neutral, positive, or purifying selection. Ka/Ks ratios (analogous to dN/dS) were also calculated by DnaSP (Rozas et al. 2003). Phylogeny of sea anemone toxins was analyzed by MEGA 3.1 (Kumar et al. 2004).

Detection of Recombination

Alignments were analyzed by recombination detection program 2 (RDP2) suite (Martin et al. 2005). As no single recombination detection model is error free or performs best in all given cases (Posada and Crandall 2001), a predicted recombination event was regarded as reliable only when identified by 2 or more of the 6 default models (RDP, GENECONV, MAXIMUM χ², BOOTSCAN, CHIMERA, and SISTER SCANNNING). All models except GENECONV use a sliding window approach in order to detect sequence incongruity (Martin et al. 2005). We used all models with their RDP2 default parameters, except for using linear sequences and a P value of 0.01.

Results

Genomic Organization of Nv1 Genes in Nematostella vectensis

The recent release of N. vectensis genome draft in the form of large scaffolds (Putnam et al. 2007; http://genome.jgi-psf.org/Nemve1/Nemve1.home.html) enabled a genomic analysis of the Nv1 genes compared with the previous release of N. vectensis genome in the form of annotated contigs (Sullivan et al. 2006; http://www.stellabase.org). This analysis revealed 14 loci correlated to the Nv1 toxin, of which 11 appear in a ∼30-kb region on scaffold 116 and 3 on scaffold 3391. Because these scaffolds still contain many sequencing gaps (fig. 1), only 8 of the loci, all on scaffold 116 and encoding for Nv1, are predicted by the genome draft to be functional genes (fig. 1). Among the Nv1 genes, very little sequence variability exists within the open reading frame and the intron (0–2.5%; up to 7/324 substitutions). A major gap of ∼10 kb in scaffold 116 could contain additional Nv1 genes (fig. 1A). Four genes are located on each side of the large gap, and no other open reading frames are found in between the Nv1 genes. All 8 genes are in the same transcriptional orientation, and their adjacent genomic sequences (up to 1 kb on each side) are highly similar but not identical (fig. 1B; table 1).

Neurotoxin Genes of 2 Other Nematostella vectensis Populations

In a previous population genetic analysis based on 153 polymorphic amplified fragment length polymorphism loci, we have shown that the Rhode River (MD), Neponset River Marsh (MA), and Crane Marsh (MA) populations are all clearly distinct from each other genetically (Reitzel et al. 2007). As Rhode River is roughly 700 km away from the 2 other habitats (fig. 2A), we verified whether the unique conservation of the Nv1 genes is restricted to the Rhode River population, which was used for the sequencing of the N. vectensis genome. Most sequences derived from individual anemones of these populations were highly similar to those of the Rhode River population with a few exceptions (fig. 2B). In the Neponset population, 4 unexpected variants were detected. Two of the clones we sequenced appear to be pseudogenes with premature stop codons and insertions that cause frameshifts (data not shown). Another 2 clones were found to bear a 6-nt deletion that changes the N-terminus of the mature toxin, creating new putative toxins named Nv3-1 and Nv3-2 (fig. 2B). Nv3-1 and Nv3-2 also differ from Nv1 at 4–5 other positions within the mature toxin region. The relative variability of nucleotide positions in all Nv1 genes was calculated for the first exon, intron, and second exon and for the regions encoding for the leader peptide and the mature toxin (fig. 2C). Notably, the first exon exhibits more variability than the intron, which in turn, exhibits more variability than the second exon, and the leader region is 2 times more variable than the mature toxin region (fig. 2C).

Av2 Neurotoxin Genes in Anemonia viridis

In order to investigate the genetic diversity of neurotoxins in other sea anemones, degenerate primers were designed based on the amino acid sequence of the mature Av2 toxin (Wunderer et al. 1976) and used to amplify the corresponding cDNA of A. viridis. The sequence of a complete Av2 transcript was obtained using 5′ and 3′ RACE. Unexpectedly, 5 variant Av2 transcripts were amplified from a single individual (supplementary fig. 1, Supplementary Material online) with the most noticeable difference at position 2 of the mature toxin, where either valine or isoleucine is found, as previously described (Wunderer et al. 1976). The variability of transcripts led us to investigate whether they are derived from one or more genes. Six different genes were amplified from 1 individual anemone: Each gene is composed of 2 exons and an intron, similar to nv1, but the av2 intron is much longer (∼1.4 kb). The genes differ from one another by single nucleotide substitutions in the intron and by indels of 50–150 bp located at 3 different sites within the intron (fig. 3A; supplementary fig. 2, Supplementary Material online). Two of these indels arise from direct tandem repeats. The regions encoding the leader and mature toxin are nearly identical in all copies. Examination by genome walking of sequences adjacent to the Av2 genes revealed that these regions were almost identical, with the only major difference being an indel of a direct tandem repeat downstream of the Av2 genes (supplementary fig. 3, Supplementary Material online).

Additional Neurotoxin Genes of Anemonia viridis

In addition to the multiple copies of av2, we amplified other sequences that may encode for novel neurotoxins using oligonucleotide primers designed for av2. One sequence (av6) was isolated from gDNA and the other 2 (av8 and av9) from mRNA. The putative toxins encoded by av6, av8, and av9 exhibit numerous substitutions and indels compared with Av2 (fig. 4B). Still, the 3 predicted proteins contain the 6 conserved cysteine residues crucial for disulfide bridging of sea anemone toxins (Honma and Shiomi 2006), bear the lysine–arginine tandem involved in cleavage of the propart (Anderluh et al. 2000), and possess a leader peptide highly similar to that of Av2 (fig. 4B). Therefore, it is likely that av6, av8, and av9 encode for active toxins. The regions encoding the mature toxin in Av2, Av6, Av8, and Av9 are so diverse at the nucleotide level that their complete multiple sequence alignment is not reliable and cannot be used for tests of selection. Nevertheless, when the most similar pair, Av2 and Av9, are aligned, tests for selection indicate that the divergence of these toxin genes was driven by positive Darwinian selection (7 synonymous vs. 24 nonsynonymous differences; Ka/Ks = 1.2).

Neurotoxin Genes in Actinia equina

Neurotoxin genes from the sea anemone, A. equina, were amplified from the gDNA of a single anemone using oligonucleotide primers designed for the mRNA encoding the toxin Ae1 (also known as AeNa; Anderluh et al. 2000). Four different genes were cloned, all sharing the structure of 2 exons separated by a variable intron exhibiting different indels. One of these genes encodes for Ae1, whereas the other 3 encode for a novel putative toxin named Ae2 (fig. 4C). Interestingly, the leader in one of these putative toxins, Ae2-2, is identical to that of Ae1, whereas the region encoding for the mature Ae2-2 is identical to that of Ae2-1 (fig. 4C). All models of RPD2 (Martin et al. 2005) that were used besides sister scanning identify a potential recombination event in the A. equina toxin genes, predicting with high probability that ae2-2 is the recombination product of ae2-1 and ae1. This prediction was corroborated using 3′ RACE on RNA isolated from a single A. equina individual. Transcripts for Ae1 and Ae2, as well as Ae3 and Ae4, in which the recombination might have occurred at the region encoding for the mature toxin, were identified (fig. 4C). To negate the possibility that such “hybrid” genes resulted from a PCR error (Bradley and Hillis 1997), the ae3 sequence was examined in 2 independent PCR amplifications. A Z-test for selection clearly indicated that ae1 and ae2 have diverged under positive selection (2 synonymous vs. 10 nonsynonymous differences; Ka/Ks = 1.6 ratio). The selection conditions of ae3 and ae4 could not be estimated because recombination might have been involved in their creation (Martin et al. 2005).

Discussion

Complete genome sequences can provide unique insights into the origin and evolution of gene families. The origin of some gene families encoding for toxins has been shown to be derived from genes that encode for nontoxic proteins. For instance, venom components in snakes appear to have been derived from multiple incidents of gene recruitment from nontoxic proteins such as liver phospholipase A2 and endothelins (Fry 2005). Another example is the recruitment of potassium channel blocker–like domains in various nontoxic proteins from cnidarians, sea urchins, and nematodes (Blaxter 1998; Moran and Gurevitz 2006). In the case of the sea anemone N. vectensis, the Nv1 genes are the only sequences that resemble Type I- and Type II-like neurotoxins (Moran and Gurevitz 2006). Although our analysis does not reveal the evolutionary roots of these toxin genes, their abundance in almost every sea anemone venom studied thus far suggests that their recruitment is ancient (Honma and Shiomi 2006). Whereas their origin remains obscure, the subsequent evolutionary history of these toxin genes appears to have been highly unusual.

Similar Patterns in nv1 and av2 Loci Suggest Concerted Evolution

As ultraconserved gene families such as those of histone and ubiquitin, previously believed to evolve through concerted evolution, were shown in recent years to actually evolve via “birth and death” evolution with strong purifying selection (Nei et al. 2000; Piontkivska et al. 2002; Nei and Rooney 2005), the conservation of Nematostella Nv1 sequences could have been attributed to selection. However, this scenario would have led to ample nucleotide variations at synonymous (silent) sites throughout the protein-coding region. This is not the case in the nv1 gene family where in most copies the coding regions, particularly those that encode the mature toxin, are practically identical at the nucleotide level, even at synonymous sites (table 1; figs. 2B and 3A). Furthermore, the fact that the 6 Av2 genes in A. viridis lack any synonymous substitutions within their exons (supplementary fig. 3, Supplementary Material online) argues that purifying selection is not responsible for the extreme conservation of these genes.

It could have been argued also that the multiple identical Nv1 genes were generated recently by a series of duplication events. After gene duplication, one of the duplicate genes would usually 1) be translocated to another genomic region, 2) become a pseudogene, and/or 3) diverge through subfunctionalization or neofunctionalization (Rodin et al. 2005). Therefore, the finding of several highly identical, functional Nv1 genes in a narrow genomic region is unusual. Pseudogenization is highly common in eukaryotes and may occur very quickly by a frameshift or nonsense mutations (Nei and Rooney 2005; Rodin et al. 2005). Nevertheless, because all nv1 copies in scaffold 116 are intact and encode for identical mature toxins, it is likely that pseudogenization and neofunctionalization of these genes did not occur. Because at least 4 duplication events were required for generating 14 Nv1 gene copies, which are unlikely to occur in a period of time too short for either neofunctionalization or especially pseudogenization of any of the Nv1 genes, the hypothesis of recent duplication is refuted.

Nematostella vectensis is a member of the Edwardsiidae, a family that is morphologically and genetically distal from most sea anemone species including A. viridis and A. equina (Daly et al. 2002; Goddard et al. 2006). Therefore, the finding of multiple Av2 genes in A. viridis with almost identical exons (fig. 3A) suggests that the conservation pattern of N. vectensis neurotoxin genes may be common to sea anemones. This suggestion is further corroborated by the identification of several genes encoding for the toxin Ae2 in A. equina (fig. 4C) and the 2 highly similar toxin genes clx-1 and clx-2 in C. parasitica (Spagnuolo et al. 1994). All toxin genes share 6 cysteine residues at conserved positions enabling similar reticulation by disulfide bridges as well as common elements in the leader and propart regions, suggesting that anemone type I and type II toxins share a common ancestor (Honma and Shiomi 2006; figs. 2B and 4A–C). Clearly, the toxin genes of each anemone species are more closely related to one another than to toxin genes of other species. Indeed, phylogenetic analysis indicates that toxins of each sea anemone species form a monophyletic group (fig. 5). This may be rationalized by assuming that either these genes were generated by recent lineage-specific duplications or that duplicate genes in the common ancestor diverged in each lineage and were homogenized via concerted evolution by gene conversion and/or unequal crossover (Eickbush TH and Eickbush DG 2007). Both options are rare in the evolution of genes, but considering the low genetic variation observed, even in introns and adjacent genomic regions (table 1), as well as the lack of pseudogenes within the nv1 gene family of the Rhode River population of N. vectensis (Nei and Rooney 2005), concerted evolution appears more likely than lineage-specific radiations.

Comparison of the Evolutionary Patterns of Nv1 and rDNA Genes

The Nv1 genes like rDNA genes, as well as their respective adjacent regions, are organized in 1 or more repetitive clusters and exhibit high sequence identity (table 1; fig. 1; Eickbush TH and Eickbush DG 2007) with slight variability mostly at nontranscribed or spliced-out regions (Gonzalez and Sylvester 2001). Like in the rDNA genes, all the Nv1 genes are organized in a uniform transcriptional orientation that differs from that of other gene families whose concerted evolution was suggested to be enhanced by their inverted orientation (Nei and Rooney 2005; Thomas 2006). Moreover, the 2 gene families are also similar in that they both exhibit different degrees of homogeneity among functional regions, for example, those that encode for the leader peptide and mature Nv1 toxin (fig. 2C). However, whereas in the genomes of many eukaryotes, including humans, rDNA gene clusters are found in different chromosomes, only 1 or 2 Nv1 gene clusters exist in the Nematostella genome. The relative position of these 2 apparent Nv1 clusters is unknown because of unsequenced gaps in the N. vectensis genome draft (fig. 1). It is noteworthy that the location of rDNA is conserved between the different chromosomes of each single species but differs between different species. This phenomenon might be the reason for the different conservation rates of nontranscribed sequences between chromosomes in humans and mice (Eickbush TH and Eickbush DG 2007). It would therefore be of interest to compare the chromosomal locations of neurotoxin genes in N. vectensis and A. viridis when their complete karyotypes become available.

Positive Darwinian Selection and Variability in Sea Anemone Toxin Genes

Despite the high intraspecific homogeneity of the Nv1 and Av2 genes, we found evidence for positive Darwinian selection acting on toxin genes in A. viridis and A. equina. The N. vectensis populations from Crane and particularly from Neponset bear toxin genes that are different from the Nv1 genes found in the sequenced genome of the Rhode River population (fig. 2B). Moreover, variability might be further enhanced by recombination as demonstrated by the Ae3 and Ae4 toxin genes of A. equina (fig. 4B). Although recombination-driven toxin variability has been proposed in cone snails (Olivera 2006), no evidence has been provided. Accelerated evolution driven by positive Darwinian selection and gene variability seems to contradict the proposed mechanism of concerted evolution. Still, variability is also found in rDNA, and several cases of rDNA that “escaped” concerted evolution are documented (Carranza et al. 1999; Keller et al. 2006; Eickbush TH and Eickbush DG 2007). Accordingly, in A. viridis and A. equina as well as the N. vectensis population at Neponset River, it is possible that some toxin genes escaped the concerted process, diverged rapidly and were fixed depending on the selective value and neutral drift of these loci. Interestingly, it was proposed that concerted evolution may increase the total rate of evolution as demonstrated for clustered nspb and nspc genes of Caenorhabditis species when compared with their paralogs that escaped the concerted process (Thomas 2006).

Concerted Evolution and Positive Darwinian Selection of Toxin Genes may Co-occur in Sea Anemones—Possible Implications

Positive Darwinian selection has been shown to act on many toxin gene families (Nakashima et al. 1993; Duda and Palumbi 1999; Zhu et al. 2004; Lynch 2007) and has been explained by the constant need for new venom compounds in a changing environment (Froy et al. 1999; Duda and Palumbi 1999; Zhu et al. 2004; Lynch 2007). Concerted evolution has been well documented only in rDNA (Nei and Rooney 2005; Eickbush TH and Eickbush DG 2007; Ganley and Kobayashi 2007). Here we suggest that this phenomenon is also common for toxin loci in various species of sea anemones. The selective advantage of concerted evolution of toxin genes may relate to a “dosage” effect of gene expression, as was also suggested for the nspb and nspc genes despite the uncertainty of their function (Thomas 2006). This suggestion is supported by a strong statistical positive correlation found between gene expression level and concerted evolution of duplicated genes in yeast (Sugino and Innan 2006). Maintaining a series of highly similar or even identical genes encoding for an abundantly expressed toxin may be advantageous due to dosage. Indeed, Av2 is a very potent toxin that accounts for 29% of the neurotoxic activity of A. viridis, is active on many sodium channel subtypes of various origins, and is also highly abundant in the venom (Beress et al. 1975; Oliveira et al. 2004; Moran et al. 2006). As Nv1 is the only toxin gene identified in N. vectensis and is encoded by a gene family that likely developed via concerted evolution, it may suggest that the dosage-related reasoning applies to this case as well.

Unlike many other poisonous animals, sea anemones lack a venom gland or a stinging organ. Instead they use specialized stinging cells, named nematocytes, found throughout their body (Kass-Simon and Scappaticci 2002). These cells are able to discharge toxins into a victim only once and then must be replaced. It is possible that this is the reason why swift production of copious toxin is advantageous in young nematocytes and the production of toxin may be accelerated by the presence of multiple copies of the same toxin gene. Although this strategy for large-scale toxin production may appear primitive or ineffective compared with the acquisition of very strong promoters and enhancers, concerted evolution may also be useful for the rapid “transmission” of advantageous mutations from a single toxin gene locus to the other loci or in preventing the loss of a highly effective toxin.

Has Concerted Evolution Occurred in Toxin Genes of Other Phyla?

If concerted evolution does play a role in the shaping of the toxin genes of sea anemones, it may serve a similar role in the development of toxin genes in other phyla. Indeed, at least 16 distinct transcripts encoding for phospholipase A2 toxins, highly similar to one another (above 95% nt identity) but different from those of other snake species, are found in the marbled sea snake Aipysurus eydouxii (Li et al. 2005). Moreover, phospholipase A2 toxin genes of another marine snake, Laticauda semifasciata, show very high identity to one another, even in introns and adjacent genomic regions (Tamiya and Fujimi 2006). In the same species, transcripts of erabutoxins, 3-finger neurotoxins, also exhibit identity of over 93% (Tamiya and Fujimi 2006). These findings suggest that toxin genes from at least 2 distinct gene families in snakes could have developed via concerted evolution. Interestingly, the mature toxin region of A. eydouxii phospholipase A2 genes is more variable than the leader peptide region (30 of 33 substitutions were nonsynonymous), indicating that positive selection had affected the evolution of these genes as well (Li et al. 2005). Noticeably, both snake species have unusual diets: A. eydouxii feeds exclusively upon fish eggs (Li et al. 2005) and L. semifasciata upon eels (Shine et al. 2002). Nematostella vectensis has a similarly restricted diet for a low diversity of invertebrates (Frank and Bleakney 1978). There is a sound reasoning for the venom of animals with specialized, narrow diets to become streamlined; however, it is also possible that concerted evolution does occur in the toxin genes of other snakes but is heavily masked by the strong positive Darwinian selection that drives the rapid diversification necessary for survival when diet is rich. Because most nucleotide data regarding toxin genes are derived from cDNAs, the distinction between identical genes or genes that vary only in their introns is impossible (e.g., Nv1 and Av2 gene copies). Therefore, more genomic data of poisonous animals are required to shed light on the impact of concerted evolution on toxin genes. It seems that identification of concerted evolution in toxin genes may have also been delayed by the plain fact that nobody was looking for it.

Conclusions

This study demonstrates the contribution of a genome project to understanding of evolutionary processes in that it provides experimental and computational findings, which strongly suggest that neurotoxin genes in sea anemones are subjected to concerted evolution. This unusual evolutionary process, which promotes homogeneity among closely related genes, has seemingly been combined with positive Darwinian selection, which promotes heterogeneity among closely related genes, in at least 2 of the sea anemone species. As more genetic, genomic, and karyotypic data accumulate for sea anemones and related anthozoan cnidarians (e.g., reef-building corals), it should become possible to determine how and to what extent concerted evolution is affecting the toxin genes of this speciose and ecologically important clade. The concerted evolution observed in nuclear rDNA, hsp70 of Drosophila and sea anemone toxin genes raises questions regarding the commonality of this phenomenon and the mechanisms involved in its maintenance and control.

We would like to thank Prof. Dan Graur of The University of Houston and 2 anonymous referees for their critical comments and helpful suggestions. M.G. was supported by the United States–Israel Binational Agricultural Research and Development grant IS-3928-06; the Israeli Science Foundation grant 909/04; and the European Community Integrated Project LSH-2005-1.2.5-2 proposal no. 037592—CONCO. J.R.F. was supported by Environmental Protection Agency grant F5E11155 (together with A.M.R.) and an National Science Foundation grant FP-91656101-0 (together with J.C.S.). A.M.R. was supported by a Postdoctoral Scholar Program at the Woods Hole Oceanographic Institution, with funding provided by The Beacon Institute for Rivers and Estuaries and the J. Seward Johnson Fund.

References

Author notes