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Published online on March 5, 2007, 10.1073/pnas.0700422104
PNAS | March 13, 2007 | vol. 104 | no. 11 | 4467-4472


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BIOLOGICAL SCIENCES / EVOLUTION
Novel sex pheromone desaturases in the genomes of corn borers generated through gene duplication and retroposon fusion

Bingye Xue*, Alejandro P. Rooney{dagger}, Masaki Kajikawa{ddagger}, Norihiro Okada{ddagger}, and Wendell L. Roelofs*,§

*Department of Entomology, New York State Agricultural Experiment Station, Cornell University, Geneva, NY 14456; {dagger}National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, Peoria, IL 61604; and {ddagger}Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259-B21 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan

Contributed by Wendell L. Roelofs, January 17, 2007 (received for review December 11, 2006)


   Abstract
Top
 Abstract
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
The biosynthesis of female moth sex pheromone blends is controlled by a number of different enzymes, many of which are encoded by members of multigene families. One such multigene family, the acyl-CoA desaturases, is composed of certain genes that function as key players in moth sex pheromone biosynthesis. Although much is known regarding the function of some of these genes, very little is known regarding how novel genes have evolved within this family and how this might impact the establishment of new sex pheromone blends within a species. We have discovered that several cryptic {Delta}11 and {Delta}14 desaturase genes exist in the genomes of the European and Asian corn borers (Ostrinia nubilalis and Ostrinia furnacalis, respectively). Furthermore, an entirely novel class of desaturase gene has arisen in the Ostrinia lineage and is derived from duplication of the {Delta}11 desaturase gene and subsequent fusion with a retroposon. Interestingly, the genes have been maintained over relatively long evolutionary time periods in corn borer genomes, and they have not been recognizably pseudogenized, suggesting that they maintain functional integrity. The existence of cryptic desaturase genes in moth genomes indicates that the evolution of moth sex pheromone desaturases in general is much more complex than previously recognized.

Ostrinia | phylogeny | pseudogene | biosynthesis | evolution

Moths possess one of the most highly developed and complex systems of long-distance mate attraction among animals. The system uses complex pheromone blends consisting of long-chain fatty acids that are often modified at the carbonyl carbon. Biosynthesis of these blends involves three key enzymatic components: uncharacterized enzymes that mediate limited chain-shortening reactions, reductases that convert acyl intermediates into alcohols, and sex pheromone desaturases that introduce double bonds at specific locations along the hydrocarbon chain. Moth sex pheromone desaturases are part of a larger family of insect acyl-CoA desaturases that originated several hundred Mya (1). So far, five distinct desaturase subfamilies have been discovered and found to function in the biosynthesis of female moth sex pheromone blends (2). These subfamilies have been named on the basis of the biochemical activities of their member genes and include the following: {Delta}9 (C16 > C18); {Delta}9 (C18 > C16); {Delta}9 (C14–C26); {Delta}10, {Delta}11, {Delta}12; and {Delta}14. One important question for researchers studying moth sex pheromones is how this unusual sex pheromone desaturase multigene family evolved and diversified.

Understanding how multigene families arise, and diversify in general, has been a subject of considerable interest over the last 40 years (3). The moth sex pheromone desaturases constitute a particularly interesting multigene family because they encode enzymes that are potential players in the establishment of barriers to reproduction and, therefore, could ultimately contribute to speciation (4). For example, the recruitment of the {Delta}14 enzyme into the female sex pheromone biosynthetic pathway of the Asian corn borer (ACB), Ostrinia furnacalis, resulted in the establishment of a novel sex pheromone blend that ultimately led to the divergence of this species from the European corn borer (ECB), Ostrinia nubilalis. Because sex pheromone desaturases are key players in moth reproduction, an understanding of the mechanisms that are responsible for generating their diversity may shed light on the molecular processes that underlie speciation in this group of insects. Thus, we initiated research to examine the diversity of sex pheromone desaturase genes and their patterns of evolution in the genomes of the ECB and ACB, two representative moth species for which a considerable amount of knowledge concerning their pheromone blends and chemical communication systems has been amassed over the past few decades (1, 4–9). Surprisingly, we found that an even larger number of "cryptic" sex pheromone desaturase genes exist within corn borer genomes and that these novel genes were generated as a result of a fusion event with a retroposon.


   Results
Top
 Abstract
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
The results of our ECB genomic library screen revealed the presence of 3 {Delta}14 gene sequences and 10 {Delta}11 desaturase genes. Two of the three {Delta}14 genes possessed nearly identical nucleotide sequences [uncorrected p distance (p) = 0.0003], but they possessed divergent upstream promoter regions (p = 0.54). The sole nucleotide difference resulted in an amino acid change at the eighth codon (excluding the start codon). A third gene was much more divergent from the other two sequences [average p distance (Formula) = 0.23] at the nucleotide level and also showed a number of amino acid changes spread throughout the deduced protein sequence. The 10 {Delta}11 desaturase genes consisted of 5 that were fully intact and 5 that were truncated. The five intact genes possessed three exons and two introns. The exon regions of two of these genes matched the published ECB {Delta}11 sequence (4). The only differences between these two sequences were found in intron 2, in which they differed in length by 87 nucleotides and diverged at an additional 73 nucleotide sites (p = 0.08). We denoted the gene containing the shorter intron as "S" and the gene containing the longer intron as "L." The other three intact sequences were very divergent from the published ECB {Delta}11 sequence (p = 0.287). One of these, which we called "ECB {psi}{Delta}11," had four nonsense mutations within the first 65 codons. The remaining five {Delta}11 genes lacked exon 1 but possessed two exons and two introns that were homologous to exons 2 and 3 and introns 1 and 2 of the intact {Delta}11 genomic sequences (Fig. 1). The truncated genes were identical to each other in these regions but possessed distinct upstream regions. In addition, they were highly divergent from the two intact genes at their homologous regions (p = 0.40).


Figure 1
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  		Fig. 1. Structure of the ezi-{Delta}11{alpha} and ezi-{Delta}11beta genes and the kaikoga element. Numbers between genes and elements indicate overall percent similarity (p distance) at the nucleotide level.

The ACB was found to possess two {Delta}14 genes and five {Delta}11 genes. The coding regions of the two {Delta}14 genes differed only on the basis of their intron 3 sequences, which were highly divergent (p = 0.478). The remaining exon/intron regions were identical. With respect to the {Delta}11 gene complement, three were fully intact, whereas the other two were truncated. Of the three intact genes, only one matched the published ACB {Delta}11 mRNA sequence. In contrast, the other two intact genes and the two truncated genes were highly divergent from the intact gene (p = 0.58, range = 0.39–0.46) and also substantially divergent from one another (p = 0.34, range = 0.25–0.51) at their exon and intron regions. One of the truncated genes, which we called "ACB {psi}{Delta}11," had two nonsense mutations within the first 20 codons. The other truncated gene was identical in structure (Fig. 1) to the ECB truncated genes in that it was missing a homologue of exon 1 but possessed exons 2 and 3 and introns 1 and 2 of the intact ACB {Delta}11 genomic sequence.

As mentioned previously, the truncated {Delta}11 gene sequences in both the ECB and ACB showed a considerable amount of nucleotide sequence divergence from the intact genomic sequences. Interestingly, the results from BLAST analyses indicated that the 5' upstream region corresponded to a reverse transcriptase (RT) ORF in the ECB truncated genes and the single ACB truncated gene that was similar in structure to the ECB truncated genes. A phylogenetic analysis revealed the ORF to be a long interspersed nuclear element (LINE) related to the RTE-1 LINE family of Caenorhabditis elegans (10) and previously unknown LINE families from the purple sea urchin Strongylocentrus purpuratus and the silkworm moth Bombyx mori (Fig. 2). The ECB–ACB LINE differed from these other LINEs by a substantial amount at the amino acid level: p = 0.63, range = 0.61–0.65 in comparison with C. elegans RTE-1; p = 0.55, range = 0.53–0.58 in comparison to the LINE from S. purpuratus; and P = 0.26, range = 0.24–0.32 in comparison with the LINE from B. mori. Thus, we have designated the ECB–ACB LINE as a new family known as ezi, which is the colloquial Mandarin Chinese word for "moth." In addition, we designated the LINE from B. mori as a different family known as kaikoga, which is the Japanese word for "silkworm moth," B. mori.


Figure 2
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  		Fig. 2. Phylogeny of cryptic and expressed {Delta}11 genes from corn borers and other representative moth species. Numbers along branches are bootstrap values; ML values are shown before the slash, and NJ values are shown after the slash. Because ML and NJ phylogenies were highly similar, only the ML phylogeny is shown.

The basic structure of the ezi-linked truncated desaturases is shown in Fig. 1. All copies except two from the ECB contain only one RT-domain. These two exceptional copies possessed one RT-domain on the plus strand and another on the minus strand (Fig. 1). In addition, the first 100 bp of the ezi-{Delta}11{alpha} element (Fig. 1) encodes an endonuclease domain. The structure of the kaikoga element is also shown in Fig. 1. This element possesses 5' and 3' UTRs as well as an endonuclease domain. We were not able to determine the number of copies of the ezi element that exist within the ECB or ACB genomes. However, multiple BLAST searches of the B. mori genome database using the B. mori kaikoga element revealed the presence of at least 200 copies within this genome, although none of the copies were associated with a sex pheromone desaturase. Similar searches of the B. mori genome database using the ezi element indicated that this family is not present in the B. mori genome.

A phylogenetic analysis of all {Delta}11 desaturase genes from both the ACB and ECB, along with the sequences from several other representative species, is shown in Fig. 3. There are four groups of corn borer {Delta}11 genes evident in this phylogeny: (i) an ezi-{Delta}11{alpha} group that is composed of genes lacking exon 1 of the fully intact {Delta}11 gene; (ii) an ezi-{Delta}11beta group that is composed of fully intact {Delta}11 genes but whose {Delta}11-homologous region is highly divergent from the "normal" {Delta}11 gene; (iii) a group consisting of the {psi}{Delta}11 pseudogene; and (iv) the normal {Delta}11 gene group. It should be pointed out that the ACB member of the ezi-{Delta}11{alpha} group lacks an ezi LINE region and contains only a {Delta}11 homologous region. This finding could have resulted if the duplication event that gave rise to this gene occurred in the common ancestor of the ECB and ACB followed by insertion of the ezi segment only in the ECB subsequent to its divergence from ACB. Alternatively, the ezi region could have been present in this gene in the common ancestor of the ECB and ACB followed by excision from the gene in the ACB subsequent to its divergence from the ECB. In light of the fact that retroposons normally do not excise themselves once they insert in a genomic location (11), this scenario seems unlikely.


Figure 3
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  		Fig. 3. Phylogeny of the ezi and kaikoga elements in relation to other LINEs. Numbers along branches are bootstrap values; ML values are shown before the slash, and NJ values are shown after the slash. Because ML and NJ phylogenies were highly similar, only the ML phylogeny is shown.

We have outlined a mechanism involving unequal crossover (Fig. 4) that explains how the ezi element became incorporated into a duplicate {Delta}11 gene and was subsequently amplified. After the ezi element was integrated into the ancestral {Delta}11 duplicate, a region of homology was established between the new ezi-{Delta}11 fusion gene and other ezi elements in other parts of the genome. Subsequent unequal crossover events occurred, resulting in amplification of the ezi-{Delta}11 fusion genes. This mode of amplification could also result, from time to time, in excision of the ezi element from certain fusion genes (as in the case of the ACB ezi-{Delta}11{alpha} gene) or incorporation of an extra ezi element into an existing fusion gene (as in the case of two ECB ezi-{Delta}11{alpha} genes) (Fig. 1). To determine when the fusion event between the ancestral {Delta}11 and the ezi LINE took place, we attempted to use a standard molecular dating method (12) based on the formula d = 2rt, in which d is the level of nucleotide sequence divergence, r is the rate of substitution, and t is the time since divergence. If we examine the level of divergence at synonymous sites by using the modified Nei–Gojobori method (13), we find that the average level (FormulaS) between the normal {Delta}11 and ezi-{Delta}11 genes is 0.927 ± 0.145, which is quite high and well above the saturation level. This considerable level of divergence at synonymous sites suggests that the ezi-{Delta}11 genes are older than the most recent common ancestor of the ECB and ACB. When we compared intact ACB vs. ECB {Delta}11 gene sequences with each other and ACB vs. ECB ezi-{Delta}11 gene sequences with each other, we found a difference in rate at synonymous sites (1.3 x 10–8 for the former and 4.1 x 10–8 for the latter assuming a date of 1 Mya for the ECB–ACB divergence). This disparity in evolutionary rate confounds attempts to date the origin of the ezi-{Delta}11 gene fusion event. Nevertheless, if we attempt to place a date by using the faster ezi-{Delta}11 gene rate, we obtain a date of 11.3 ± 1.8 Mya. The use of the slower {Delta}11 rate produces a date of 35.7 ± 5.6 Mya. Alternatively, if we assume that the rate of synonymous site variation among nuclear genes in Drosophila (15.6 substitutions per site per 109 years) (14) is about the same as it is in the ECB and ACB, we can use this rate to obtain a date of 29.7 ± 4.7 Mya, which is closer to the {Delta}11 rate. When nucleotide sites have reached the saturation level, it is better to use the deduced amino acid sequence for molecular dating because it evolves more slowly (15). There are no amino acid substitutions between the normal {Delta}11 genes of the ECB and ACB, but there are several between the ECB and ACB ezi-{Delta}11 genes. The rate of deduced amino acid substitution between these genes is 1.8 x 10–8. If we use this rate along with the Poisson-corrected amino acid distance estimate (16) between the normal {Delta}11 and ezi-{Delta}11 genes (P = 0.305 ± 0.041), we obtain a corresponding date of 8.5 ± 1.1 Mya, which is close to the date of 11.3 ± 1.8 Mya calculated by using the faster ezi-{Delta}11 gene rate.


Figure 4
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  		Fig. 4. Proposed genomic mechanism involving unequal crossover, duplication, and amplification to explain the origin of ezi-{Delta}11 genes in the ECB and ACB genomes. The same color legend represented in Fig. 1 applies here.

Finally, we were interested in the probability that the ezi-{Delta}11 genes retain their functionality. If these genes had reverted to a nonfunctional state, we would have seen evidence of a shift toward neutral evolution in which dS would not be statistically different from the levels of nonsynonymous substitutions per site dN. However, the difference in magnitude between dS and dN is consistent with a pattern of purifying selection because the results of statistical tests (Z-test and Fisher's exact test) were significant for dS > dN in all comparisons between the ACB and ECB ezi-{Delta}11 gene duplicates themselves as well as between them and the normal ACB and ECB {Delta}11 genes. To examine this in more detail, we conducted several simulation analyses aimed at examining the process of pseudogenization. In our simulations, we found that t1/2 (the time required for an intact ORF to be interrupted in half of the simulation replications) to be 0.12 million years by using the inferred ezi-{Delta}11 substitution rate. The resultant probability that an ezi-{Delta}11 gene retains its ORF was thus found to be 4.5 x 10–29, assuming a divergence date of 11.1 Mya, or 3.1 x 10–75, assuming a divergence date of 29.7 Mya. If we use the Drosophila substitution rate, we obtain t1/2 = 0.35 million years, which gives a probability of 2.9 x 10–10, assuming a divergence date of 11.1 Mya, or 2.8 x 10–26, assuming a divergence date of 29.7 Mya. Because the ORFs have remained intact in the face of such a small probability that they would not, under a model of neutral evolution, they must be subject to purifying selection. Similarly, in our simulations designed to test whether the observed number of frameshift mutations is equal to or less than what would be expected by chance, we found that ezi-{Delta}11 functional integrity is probably maintained (Pdis < 0.001). We also simulated whether the ratio of the observed number of nonsynonymous (NA) and synonymous (NS) substitutions deviates from the neutral expectation over the evolutionary time frames inferred above and found that the result was highly significant in either case (PNa/Ns < 0.001).


   Discussion
Top
 Abstract
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
In previous studies (1, 4), we suggested that sex pheromone desaturase genes undergo birth-and-death evolution on the basis of clustering patterns evident in the sex pheromone desaturase phylogeny. Although certain exceptions exist (17), a multigene family that undergoes birth-and-death evolution will typically include some members that are classical pseudogenes (i.e., nonfunctionalized genes that possess frameshift mutations and/or premature stop codons in their reading frames) (3). In this study, we found evidence for the presence of one classical {Delta}11 pseudogene in both the ACB and ECB genome, the {psi}{Delta}11 gene (Fig. 3). Yet, the more interesting findings concern the presence of several duplicate {Delta}11 and {Delta}14 genes in both the ECB and ACB genomes.

Elucidating the mechanisms by which new genes originate and gain new function(s) has been a subject of intense interest in the study of multigene families and evolutionary genomics over the past several decades (3, 12, 14, 18, 19). The recruitment of transposable elements into gene duplicates is believed to be one mechanism by which gene duplicates can evolve new functions (20, 21). This process has been shown to play a role in the generation of new genes in a variety of animal and plant taxa (20–26). One way that this type of cooption occurs is through fusion or chimerism in which the mobile element is incorporated into a gene duplicate and the chimeric gene subsequently takes on a new function if it survives in the genome. In this study, we have identified several genes in the ECB and ACB that were derived from the fusion of a LINE with a {Delta}11 sex pheromone desaturase gene. The LINE, which we call ezi, represents a novel family of retroposons related to the RTE-1 family in C. elegans (10).

To explain the origin these fusion genes, we hypothesize that a {Delta}11 gene was duplicated in an ancestor of the ECB and ACB. Our molecular dating analyses suggest that this event took place some time during the Miocene. In light of our results, we feel confident in saying that most, if not all, members of the genus Ostrinia should possess ezi-{Delta}11 genes. However, it is less clear whether other members in the same subfamily (Pyraustinae) or family (Crambidae) will also possess these genes. Obviously, future laboratory investigations will be required to answer this question. After this initial duplication event occurred, there appear to have been two distinct fusion events: one for the ezi-{Delta}11{alpha} genes and one for the ezi-{Delta}11beta genes. However, the ezi element was integrated into different positions within the desaturase-homologous region of the ezi-{Delta}11{alpha} gene (i.e., in intron 1) versus the ezi-{Delta}11beta gene (i.e., upstream of exon 1) (Fig. 1). After these integrations took place, unequal crossover resulted in amplification of the new ezi-{Delta}11 fusion genes (Fig. 4). It should be pointed out that the occurrence of independent integrations in the ezi-{Delta}11{alpha} versus ezi-{Delta}11beta genes suggests that there is a "signal" sequence in the 5' region of the normal {Delta}11 gene that facilitates the insertion of retroposons. If this is the case, it could be that more LINE-desaturase fusion genes exist in the genomes of other moth species. An important question is whether the ezi-{Delta}11 gene duplicates are pseudogenes.

We did not find any evidence to suggest that the ezi-{Delta}11 genes in the ECB and ACB are classical pseudogenes because their reading frames are intact. Of course, other mechanisms of pseudogenization are possible (e.g., promoter nonfunctionalization). Yet, if ECB and ACB are classical pseudogenes, it is surprising that the ezi-{Delta}11 gene duplicates have remained intact for at least over 1 million years since the divergence of the ECB and ACB. One would have expected some sort of frameshift or nonsense mutation to have occurred in the reading frames of these genes over the course of this time period. In contrast, our analyses strongly suggest that these genes have retained functionality and have been subjected to purifying selection during their evolution. Thus, the function(s) of these genes and whether or not they influence sex pheromone biosynthesis should be investigated. Such studies would provide an interesting opportunity to learn how LINEs contribute to the evolution of novel gene sequences.

What, then, are the implications concerning these ezi-{Delta}11 fusion genes as well as the other cryptic {Delta}11 and {Delta}14 genes in corn borer genomes? Certainly, the existence of cryptic sex pheromone desaturases in moth genomes indicates that their evolution is much more complex than previously recognized. In addition, if these genes are functional, or possess the capacity to become functional, they could potentially serve as raw material from which new pheromone blends could arise if the genes were coopted into sex pheromone biosynthesis pathways. Of course, it is entirely possible that these genes do not function in sex pheromone biosynthesis at all, and they may have been coopted to perform some other unrelated function. Exploration of these possibilities would provide important insights into how novel gene functions arise in genomes in general and, in this particular case, how it affects the process of mate attraction in moths.


   Materials and Methods
Top
 Abstract
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
GenomeWalker (Clontech, Mountain View, CA) genomic DNA libraries were constructed from DNA isolated from (i) a laboratory population of the ECB Z-strain maintained at the New York State Agricultural Experiment Station and (ii) a Korean laboratory population of the ACB. Previously published (2) degenerate primers that target the conserved central region of moth desaturase genes were used to clone the genomic sequences by using genomic DNA as a template. Once desaturase-homologous sequences were identified, the full-length genomic DNA sequence for each clone was subsequently obtained following a primer walking strategy.

DNA sequences from all clones were edited by using Lasergene sequence analysis software (DNASTAR, Madison, WI) with minor editing after visual inspection. The edited sequences were then used to query both the entire GenBank database and the complete genome databases of B. mori, Aedes aegypti, Anopheles gambiae, and Drosophila melanogaster by using BLAST searches targeting both nucleotide and protein sequences. The nucleotide sequences for the closest matches for each of the clones were compiled into an alignment with the nucleotide sequences of the ECB and ACB desaturase clones. The alignment was created with the computer program CLUSTAL-X (27), followed by visual inspection and manual adjustment.

Phylogenetic analyses were conducted by using the maximum likelihood (ML) and neighbor-joining (NJ) methods. The former were conducted by using the computer program PHYLIP 3.65 (28), and the latter were conducted by using the computer program MEGA 3.1 (29). For phylogenetic analysis of {Delta}11 plus ezi-{Delta}11 sequences, trees were constructed from nucleotide sequences by using the F84 + {gamma} model for ML analyses and the Tamura-Nei + {gamma} model for NJ analyses. In both cases, the {gamma} shape parameter ({alpha}) was 0.7, which was estimated by using the maximum likelihood method (16). For phylogenetic analysis of LINE sequences, trees were constructed from protein sequences by using the Jones–Taylor–Thornton (30) model for ML analysis and the Poisson model (16) for NJ analysis. The statistical reliability of internal branches was assessed by using 1,000 bootstrap pseudoreplicates for ML analyses and 1,500 pseudoreplicates for NJ analyses.

The method of Zhang and Webb (31), as implemented in the program PSEUDOGENE (31), was used to compute the rate at which an ORF becomes disrupted by using both the inferred ezi-{Delta}11 substitution rate and the Drosophila synonymous substitution rate (14). We assumed that the insertion–deletion rate was 15% of this estimate (32). By evaluating the magnitude of the probability that an ezi-{Delta}11 gene retains an intact ORF in relation to the divergence time, we then gauged the relative likelihood that the gene has remained subject to selective constraints. The method of Dupanloup and Kaessmann (33), as implemented in the program ReEVOLVER 1.0 (33) was used to compute the probability (Pdis) that the observed number of frameshift mutations is equal to or less than what would be expected by chance and the probability (PNa/Ns) that the ratio of NA to NS deviates from the neutral expectation. Both of these probabilities are computed through comparison of the observed value with the frequency distribution generated through simulation. The simulation requires the reconstruction of ancestral sequences, which we conducted by using the likelihood method (34) as implemented in ReEVOLVER. We used the inferred ezi-{Delta}11 substitution rate and assumed that the insertion–deletion rate was 15% of this estimate.


   Acknowledgements
Top
 Abstract
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Dr. Jin Kyo Jung (National Institute of Crop Science, Suwon, Korea) for providing ACB pupae from his laboratory culture, Drs. Jianzhi Zhang and Robert Friedman for providing helpful comments, and Dr. Isabelle Dupanloup for help with the program ReEVOLVER. This study was supported by U.S. Department of Agriculture National Research Initiative Competitive Grants Program Grant 2002-35302-12287 (to W.L.R.).


   Footnotes
 

Abbreviations: ACB, Asian corn borer; ECB, European corn borer; LINE, long interspersed nuclear element; ML, maximum likelihood; NJ, neighbor-joining.

§To whom correspondence should be addressed. E-mail: wlr1{at}cornell.edu

Author contributions: B.X. and W.L.R. designed research; B.X. performed research; B.X., A.P.R., M.K., and N.O. analyzed data; and B.X., A.P.R., and W.L.R. wrote the paper.

The authors declare no conflict of interest.

Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. EF113390–EF113404 and EF125923–EF125927).

© 2007 by The National Academy of Sciences of the USA


   References
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 Abstract
 Results
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 Materials and Methods
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