Molecular Biology and Genetics, Cornell University
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Abstract |
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Key Words: Drosophila melanogaster antibacterial peptide genes polymorphisms propeptide domains, innate immunity
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Introduction |
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One major model of host-pathogen evolution driven by natural selection is the coevolutionary "arms race" (Dawkins and Krebs 1979). The premise of this model is that pathogens continually evolve to defeat host defenses, while the host continually evolves novel means of pathogen suppression. Therefore, new virulence and resistance alleles sequentially sweep through pathogen and host populations. The model posits that the host population should be in a continual state of recovery from selective sweep, so a generally low level of standing genetic variation is predicted. The expected degree of depression of variation depends on the intensity of selection and the frequency of favorable mutations (Wiehe and Stephan 1993). However, if the process is truly a "race," the sweep events must be fairly common, even overlapping, with the selected amino acids frequently fixing in the population. If insect antimicrobial peptides evolve according to the arms race model, their genes should show elevated amino acid differentiation and low levels of standing variation, with indications of rapid and frequent allelic turnover via strong directional selection.
Under a second model, natural selection may favor genetic variability in a host population either if rare alleles are favored by virtue of their rarity or if variability in the host locus confers resistance to multiple distinct pathogens. A classic example of hypervariability generated by Darwinian selection is provided by the antigen recognition site of the vertebrate major histocompatibility complex (MHC) locus (Hughes and Nei 1988). Were insect antimicrobial peptide genes to conform to the hypervariability model, they would be expected to harbor substantial levels of amino acid polymorphism, perhaps exceeding even the level of silent variation in coding regions.
A third hypothesis is that insect antibacterial peptides may conform to the neutral model of molecular evolution (Kimura 1983). Under this model, the vast majority of mutations are sufficiently deleterious that they are rapidly removed from the population. The empirically observed mutations are thus neither favored nor disfavored by natural selection. Extensive theoretical work on this model makes it valuable as a null hypothesis, and there is some a priori evidence that D. melanogaster antibacterial peptides may evolve more or less neutrally. Prior surveys of natural variation in D. melanogaster cecropin and diptericin A genes have not detected marked departures from the neutral expectation (Clark and Wang 1997; Date et al. 1998; Ramos-Onsins and Aguadé 1998). The consistent and widespread observation of highly conserved antibacterial peptide sequences across vast evolutionary distances (Boman 1995; Bulet et al. 1999) further argues against rapid, adaptive amino acid substitution as a general model of antibacterial peptide evolution.
This study revisits previously published surveys of natural variation in the attacin C (Lazzaro and Clark 2001), cecropin A1, A2, B, and C, and diptericin A (Clark and Wang 1997) genes and adds polymorphism and divergence data from the single-copy loci defensin, drosocin, and metchnikowin. The data from these genes are assembled and examined for systematic departures from a neutral evolutionary process. In particular, high rates of amino acid substitution and skews in the distribution of allele frequencies at polymorphic sites may be signatures of natural selection. It is likely that the pathogens D. melanogaster faces in North America are distinct from those found in sub-Saharan Africa, and because previous work has found significant population differentiation among alleles of the diptericin A and cecropin genes (Clark and Wang 1997) we focus here exclusively on North American alleles.
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Materials and Methods |
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New sequences were obtained for the defensin, drosocin, and metchnikowin loci. Sequence data were collected from the same 12 D. melanogaster lines and the same D. simulans line surveyed in Lazzaro and Clark (2001). Oligonucleotide primers for the defensin, drosocin, and metchnikowin genes were designed based on GenBank accession numbers Z27247, X98416, and AF030959. Primer sequences are available upon request. The survey region for defensin begins 1,123 bp upstream of the translational start codon, includes the entire 279 bp of coding sequence, and terminates 3 bp downstream of the stop codon. The drosocin region begins 933 bp upstream of translational start and continues to the end of the 195-bp coding region. The antibacterial peptide gene attacin A begins 1.2 kb downstream of the drosocin gene. Polymorphism and divergence in the sequence between drosocin and attacin A was described by Lazzaro and Clark (2001) and is qualitatively and quantitatively similar to the drosocin survey region described here. The metchnikowin survey region begins 1,499 bp upstream of the start codon, reads through the 159-bp coding sequence, and terminates 106 bp 3' of the stop codon. defensin, drosocin and metchnikowin are all intronless. PCR-amplified templates were directly sequenced on either an Applied Biosystems 373 or a Beckman Coulter CEQ2000 automated sequencer, using modifications of the manufacturers' suggested protocols. All sequences were verified on both strands. The defensin, drosocin, and metchnikowin sequences have been deposited in GenBank under accession numbers AY224604 to AY224642.
Statistical Analysis
Sites with alignment gaps were excluded from all statistical analyses of nucleotide polymorphism and divergence data. Four sites (three in drosocin and one in cecropin C) where three nucleotides are segregating within D. melanogaster were also excluded. At all other polymorphic sites, the parsimonious assumptions were made that the state of the D. simulans allele reflects the ancestral state of the polymorphism and that the probability of back-mutation within D. melanogaster is negligible. Eleven sites where D. simulans has a third nucleotide, different from either state of a D. melanogaster polymorphism, were excluded from analyses that make use of outgroup information. Polymorphic sites tables for diptericin A, the cecropins, and attacin C can be found in Clark and Wang (1997) and Lazzaro and Clark (2001). Supplemental figures 13 for this manuscript show polymorphic sites and fixed differences observed in defensin, drosocin, and metchnikowin (see online Supplementary Material).
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Because extant North American D. melanogaster are believed to be derived from an ancestral African population (David and Capy 1988), we tested the empirically observed data against simple null models of population founding followed by expansion. These were approximated by simulating equilibrium neutral populations maintaining effective size N0 for 4N0 generations, then introducing a single bottleneck of varying severity at various times before present. In all cases, the bottleneck was maintained for 0.0001 x N0 (approximately 100 in D. melanogaster) generations, after which the population was allowed to grow to a size of 0.1 x N0. Five parameter combinations were tested, with 10,000 genealogies simulated under each parameter set. In three cases, the bottleneck was set to have occurred 0.002 x N0 (approximately 2,000) generations before present, and the population size was reduced to either 0.001 x N0 (approximately 1,000), 0.0001 x N0 (approximately 100), or 0.00001 x N0 (approximately 10) individuals during the bottleneck phase. In two additional cases, the severity of the bottleneck was set to 0.0001 x N0, the value under which the empirical data had the highest probability in the first sets of simulations, but the age of bottleneck was set to either 0.0005 x N0 (approximately 500) or 0.05 x N0 (approximately ) generations before present. The conservative assumption of no recombination is made in all simulations incorporating a demographic component. Varying the recombination rate had substantial effect only after a very ancient bottleneck (2 x N0 generations before present), and in this scenario simulations were similar to those assuming no demographic structure (data not shown). In no case was migration between the founded and ancestral population simulated.
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Results |
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There is one position each in diptericin A and cecropin C with three amino acid residues segregating. At both sites, the two mutations in D. melanogaster are nonconservative with respect to the ancestral state inferred from D. mauritiana. The three-state position in the cecropin C signal peptide has additionally mutated a fourth time in the history of Drosophila cecropin genes, as this residue is fixed for a nonconservative replacement between cecropins A1 and A2 and cecropins B and C (fig. 1). Another cecropin residue shows evidence of convergent multiple mutations in the C-terminal portion of cecropins B and C, where an AlaGly mutation appears to have occurred independently in homologous positions of D. melanogaster cecropin B and D. mauritiana cecropin C (fig. 1). The ancestral state at this position is Ala in both loci, as determined from sequences obtained from GenBank of D. simulans (Y16860 [Ramos-Onsins and Aguadé 1998] and AB010790 [Date et al. 1998]), D. yakuba, D. teissieri, D. orena, D. erecta, D. takahashii (AB047059 to AB047063 [Date-Ito et al. 2001]) and D. virilis (U71249 [Zhou, Nguyen, and Kimbrell 1997]).
The nearly significant excess of amino acid polymorphisms relative to replacement fixations observed in the peptide data could potentially reflect a low level of purifying selection if the polymorphisms are nearly neutral or slightly deleterious, particularly since most of the polymorphic sites are in processed domains that may experience little functional constraint. If this is the case, then the allele frequency spectrum of polymorphic sites should resemble that predicted under selective neutrality. Derived mutations at high frequency are rare under a neutral process but may be more common if neutral polymorphisms "hitchhike" to high frequency when nearby sites are selectively favored (Fay and Wu 2000), particularly during and soon after the selective event (Przeworski 2002). Antibacterial peptide genes tend to contain an excess of high-frequency derived sites, measured by Fay and Wu's H (table 3). This tendency is especially pronounced when critical values are determined assuming a population recombination rate equivalent to meiotic recombination rates observed in the laboratory (Fisher's combined probability, ) but is apparent even under the conservative assumption of no recombination (Fisher's combined probability,
).
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Because demography should have genome-wide effects, comparisons between the peptide loci and functionally unrelated D. melanogaster genes can reveal whether population structure causes the observed departures of H values from the null expectation. Andolfatto and Przeworski (2001) have assembled polymorphism data from D. melanogaster loci previously surveyed by other authors. We subsampled their data, retaining only North American alleles from each locus when five or more North American alleles were sampled and were segregating for five or more polymorphic sites. H was calculated for each of these 12 loci (Acp26A, est6, G6PD, hsp83, mlc1, per, pgd, ref(2)p, SOD, tpi, v, and w), and the probability of observing an H as or more negative than that observed was determined by simulation assuming panmixia and no recombination. The combined probability for the Andolfatto and Przeworski loci is marginally significant (,
), although less so than the combined probability of the observed peptide data (
,
). When two nonpeptide loci that have individually significant negative H values (vermilion,
; white,
) are excluded, the combined probability across the genome sample is nonsignificant (
,
). When the single peptide locus with an individually significant H is excluded (Diptericin A,
), the combined probability of the remaining peptide loci is still nearly significant (
,
).
Excepting the cecropins, the antibacterial peptide genes tend to show an excess of linkage disequilibrium, with estimated from the data (Hudson 1987) ranging from two to four orders of magnitude smaller than 4
r calculated from the laboratory meiotic recombination rate (table 1). The amount of linkage disequilibrium in a data set can be measured using ZnS, a statistic based on the sum of coefficients of linkage disequilibrium across all pairs of sites in the sample (Kelly 1997). As expected, there is no evidence of excess linkage disequilibrium in any of the antibacterial peptide genes when critical values of ZnS are determined by simulations assuming either no recombination or a recombination rate equal to the empirically estimated C. More extreme P-values are observed, however, when the null distribution of ZnS is determined using the recombination parameter estimated from laboratory recombination rates (Fisher's combined probability
[table 4]), indicating that the antibacterial peptide genes have an overall excess of linkage disequilibrium, given our best estimates of their actual meiotic recombinational environments. Excess linkage disequilibrium can be generated by natural selection or under numerous demographic scenarios, although the effect of positive selection on disequilibrium is expected to be short-lived (Przeworski 2002). The cecropin genes differ from the remainder of the peptide genes in that
is approximately equal to 4
r in cecropins A1 and A2, and
is greater than 4
r in cecropins B and C. Gene function, genome arrangement, and other possible explanations for this discrepancy are considered in the Discussion.
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The drosocin locus has one of the most extreme values of ZnS and of H among the peptide genes. There are only two amino acid polymorphisms and no fixed differences in drosocin, one of the polymorphisms being a polarity-changing Ala/Thr polymorphism at intermediate frequency in the propeptide domain (fig. 1). A preliminary analysis suggested that the Ala alleles were deficient in polymorphism compared with the Thr alleles, although Ala is inferred to be the ancestral state. We pursued this observation by sequencing approximately 205 bp upstream and 357 bp downstream of the Ala/Thr site in an additional 20 chromosomes collected in Pennsylvania, USA, in 2001 (these sequences have been deposited into GenBank under accession numbers AY224643 to AY224662). Surprisingly, only three of these additional lines had Thr at the variable position, although seven of the original 12 alleles encoded Thr alleles. It is significantly unlikely that these two samples (7 Thr:5 Ala and 3 Thr:17 Ala) were taken from populations with the same allele frequency (,
), raising the possibility that the Ala allele might have substantially increased in frequency in only two years.
Twenty-one of the 22 Ala alleles cluster in a single clade distinct from the Thr alleles. The remaining Ala allele creates the most basal D. melanogaster branch in the entire genealogy when the tree is rooted with the D. simulans sequence (fig. 2). Only seven sites are segregating within the 21-allele internal Ala clade. Six of these are unique to that clade, and none have a frequency higher than 0.095 within the clade. In contrast, there are 21 sites segregating among the 10 Thr alleles (fig. 3). Despite the small number of sites, Tajima's D statistic (Tajima 1989) is significantly negative within the internal Ala clade, indicating a significant excess of rare polymorphisms (,
; all simulations in this short sequence window assume no recombination). Among the Thr alleles only,
(
). Inclusion of the basal Ala allele with those in the internal Ala clade adds several more rare polymorphisms (
,
) and results in a highly significant excess of high-frequency derived mutations (
,
). With the exclusion of the basal Ala allele, however, the common derived sites are no longer polymorphic but become fixed differences relative to D. simulans, resulting in nonnegative value of H (
,
) and illustrating a peculiarity of the H test. H is significantly negative over the entire 32 allele data set (
,
) but not among Thr alleles alone (
,
). Overall, these data are consistent with a model of positive selection driving the expansion in frequency of the internal Ala clade. D. simulans and D. yakuba (not shown) sequences both indicate that Ala is the ancestral state at this residue, suggesting that this position may have mutated from Ala to Thr early in the history of the D. melanogaster lineage and then mutated back to Ala in the expanding clade (fig. 2). If the Ala/Thr polymorphism is itself the target of selection, this history implies that selection pressure has changed over time. The presence of the basal Ala allele also complicates the interpretation that the Ala/Thr polymorphism is the target of selection, although this allele may be a recombinant. Alternatively, selection may be acting not on the Thr/Ala polymorphism, but rather on a site linked to the high-frequency Ala allele.
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Discussion |
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Neither do the data support the classical model of selectively maintained hypervariability in mature antibacterial peptides, as the rate of silent substitution is higher than the nonsynonymous rate. Nevertheless, these genes have a high level of amino acid polymorphism relative to interspecific divergence (table 2), with most amino acid variation located in domains that are proteolytically removed to activate the peptide (fig. 1). Several of these polymorphisms are radical, changing charge or polarity at the variable residue. This could be attributable to an absence of purifying selection, making the observed polymorphisms effectively neutral, although in that case, a higher proportion of the amino acid substitutions might be expected to drift to fixation. Alternatively, the segregating amino acid polymorphisms might be slightly deleterious, preventing their fixation, although deleterious mutations are rarely expected to achieve intermediate frequency as several of the peptide polymorphisms do (fig. 1). The nearly significant excess of amino acid polymorphism relative to fixation might be achieved if rare amino acid variants are selectively favored, but only when rare, and if selective advantage is lost as the variant becomes more common. If this is true, the peptide genes could be expected to show other indications of selection. Some evidence of that positive selection affects allele frequencies is provided by the general excess of high-frequency derived mutations and the high degree of linkage disequilibrium in the peptide genes. Potential selection is most clearly illustrated at the drosocin locus, where one allele seems to have recently and rapidly increased in frequency.
The genes showing the strongest departure from the equilibrium null model (drosocin, metchnikowin, and diptericin A) are completely unlinked, being distributed across 4.1 Mbp of chromosome 2. The only naturally occurring inversion polymorphism of appreciable frequency in this chromosomal region is In(2R)NS. Both drosocin and metchnikowin are outside the breakpoints of this inversion, and independent genetic evidence (unpublished data) suggests that the 12 alleles sequenced at these loci are all of the standard arrangement. The cytological arrangements of the 12 diptericin A alleles, which are inside In(2R)NS, are not known. It is possible that In(2R)NS polymorphism within the sample could affect estimates of /4
r and ZnS, but inversion polymorphism alone is not expected to affect H. We therefore find it unlikely that inversion polymorphism underlies the observed data.
It is important to note that skewing of the site frequency spectrum and departure from linkage equilibrium can have demographic causes as well as selective ones. Przeworski (2002) has shown that an extreme degree of population subdivision with unequal sampling across subpopulations can give a significant departure of H from the neutral expectation, but, as acknowledged by Przeworski, such a model is not likely to represent real Drosophila populations. Our simulations demonstrate that a more plausible model of population bottleneck followed by expansion can also generate values of H reflecting an excess of high-frequency derived polymorphisms. One way of distinguishing demographic from selective effects is by comparison with other loci in the genome. The departure of the pooled peptide data from neutral panmictic expectations is qualitatively similar to, although more extreme than, that of the pooled genome-wide data in terms of linkage disequilibrium and skew in site frequency spectrum. Even this comparison, however, is problematic because of variability in the power to detect departure from the null among loci due to differences in number of alleles surveyed and polymorphic sites observed. Furthermore, far from being randomly chosen, the genome-wide "control" loci are subject to both experimenter and publication bias. This complication is illustrated by the fact that the two loci that drive the marginal combined probability significance of H in the genome-wide data, vermilion (Begun and Aquadro 1995) and white (Kirby and Stephan 1995), were surveyed in anticipation of detecting natural selection. Additionally, Fay, Wyckoff, and Wu (2002) have used the Andolfatto and Przeworski (2000) data to argue that natural selection is pervasive in the D. melanogaster genome. A convincing distinction between selective and demographic effects would require comparison to polymorphism data from loci sampled throughout the genome without regard to expected selective history, and such a control data set does not currently exist for D. melanogaster.
The cecropin genes, in particular cecropin C, differ noticeably from the remainder of the genes in their comparative lack of both linkage disequilibrium (tables 1 and 4) and skew in the site frequency spectrum towards common-derived variants (table 3). The contrasting patterns of variability may reflect functional differences among the genes. While the rest of the genes are induced by larvae and adults in response to systemic infection, cecropins B and C are expressed in pupae during metamorphosis, where they may be exposed to less pathogenic or variable bacteria, for instance those residing in the larval gut. This interpretation is consistent with the observation that cecropins B and C are more similar to each other at the amino acid level than either is to cecropin A1 and A2 (fig. 1). The departure of the cecropins from the remainder of the antibacterial peptides may be partially attributable to genomic arrangement, as well. Cecropins A1, A2, and B are within a 4-kb segment of chromosome 3R, with cecropin C less than 4 kb away. With this in mind, it might be better to consider the Cecropin genes as a single superlocus with respect to recombination and H. However, inconsistencies in sample size and composition from gene to gene within the cecropin cluster (Clark and Wang 1997) preclude their concatenation into a single data set. The treatment of the tightly linked cecropin genes as separate loci may be justified by the fact that these genes show the lowest levels of intragenic linkage disequilibrium.
Individual data from metchnikowin, diptericin A, and drosocin and combined data from all of the peptide loci suggest the effects of natural selection in the recent past, although demographic history is also likely to have played a role in the evolution of these genes. We observed virtually no amino acid differentiation between species and little amino acid polymorphism in the mature peptide domains. If selection is acting on these loci it likely acts either on regulatory variants or on the substantial number of nonconservative amino acid polymorphisms in proteolytically processed domains. Selection might favor such polymorphisms if they provide protection against immunomodulatory molecules injected by pathogenic bacteria into the host cell. Bacterial injection of proteins that interfere with host cell signaling pathways and immune responses have been well documented in plants and animals (Hueck 1998; Cornelis and Van Gijsegem 2000; Ernst 2000). Data from an immunity-related Drosophila transcription factor, Relish, conceptually supports the bacterial interference model. Relish proteins have an autoinhibitory domain, which is proteolytically cleaved to activate the transcription factor (Dushay, sling, and Hultmark 1996). The amino acids surrounding the site of Relish cleavage have an extremely high rate of amino acid substitution (Begun and Whitley 2000b), suggesting that these amino acids may also be an intracellular site of host-pathogen coevolution. The Relish data, however, document rapid fixation of amino acid substitutions, whereas the peptide genes evolve very slowly. Amino acid variation could conceivably be maintained with no increase in the rate of fixation if selection is dependent on the allele frequency of the targeted site. More direct experiments on pathogen-Drosophila biology are obviously required to test this hypothesis.
At present, the data allow the firm rejection of arms races and maintained hypervariablility as appropriate models to describe the evolution of antibiotically active domains of Drosophila antibacterial peptides. But there is suggestive evidence that natural selection may act on these genes, perhaps favoring radical amino acid variability in a frequency-dependent manner and in response to pressure from pathogens. Further research is necessary to test this model and to conclusively separate demographic from selective effects.
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Acknowledgements |
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Footnotes |
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E-mail: brian.lazzaro{at}cornell.edu.
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Andolfatto, P., M. Przeworski. 2000. A genome-wide departure from the standard neutral model in natural populations of Drosophila. Genetics 156:257-268.
Åsling, B., M. S. Dushay, and D. Hultmark. 1995. Identification of early genes in the Drosophila immune response by PCR-based differential display: the attacin A gene and the evolution of attacin-like proteins. Insect Biochem. Mol. Biol. 25:511-518.[CrossRef][ISI][Medline]
Begun, D. J., and C. F. Aquadro. 1995. Molecular variation at the vermilion locus in geographically diverse populations of Drosophila melanogaster and D. simulans. Genetics 140:1019-32.
Begun, D. J., and P. Whitley. 2000a. Reduced X-linked nucleotide polymorphism in Drosophila simulans. Proc. Natl. Acad. Sci. USA 97:5960-5965.
Begun, D. J., and P. Whitley. 2000b. Adaptive evolution of Relish, a Drosophila NF-B/I
B protein. Genetics 154:1231-1238.
Boman, H. G. 1995. Peptide antibiotics and their role in innate immunity. Annu. Rev. Immunol. 13:61-92.[CrossRef][ISI][Medline]
Bulet, P., J.-L. Dimarcq, C. Hetru, M. Lagueux, M. Charlet, G. Hegy, and A. Van Dorsselaer. 1993. A novel inducible antibacterial peptide of Drosophila carries an O-glycosylated substitution. J. Biol. Chem. 268:14893-14897.
Bulet, P., C. Hetru, J.-L. Dimarcq, and D. Hoffmann. 1999. Antimicrobial peptides in insects; structure and function. Dev. Comp. Immunol. 23:329-344.[CrossRef][ISI][Medline]
Carvalho, A. B., and A. G. Clark. 1999. Intron size and natural selection. Nature 401:343-344.[CrossRef][ISI][Medline]
Clark, A. G., and L. Wang. 1997. Molecular population genetics of Drosophila immune system genes. Genetics 147:713-724.
Cornelis, G. R., and F. Van Gijsegem. 2000. Assembly and function of type III secretory systems. Annu. Rev. Microbiol. 54:735-774.[CrossRef][ISI][Medline]
Date, A., Y. Satta, N. Takahata, and S. I. Chigusa. 1998. Evolutionary history and mechanism of the Drosophila cecropin gene family. Immunogenetics 47:417-429.[CrossRef][ISI][Medline]
Date-Ito, A., K. Kasahara, H. Sawai, and S. I. Chigusa. 2002. Rapid evolution of the male-specific antibacterial protein andropin gene in Drosophila. J. Mol. Evol. 54:665-670.[CrossRef][ISI][Medline]
David, J. R., and P. Capy. 1988. Genetic variation of Drosophila melanogaster natural populations. Trends Genet. 4:106-111.[CrossRef][ISI][Medline]
Dawkins, R., and J. R. Krebs. 1979. Arms races between and within species. Proc. R. Soc. Lond. B Biol. Sci. 205:489-511.[ISI][Medline]
De Gregorio, E., P. T. Spellman, G. M. Rubin, and B. Lemaitre. 2001. Genome-wide analysis of the Drosophila immune response by using oligonucleotide microarrays. Proc. Natl. Acad. Sci. USA 98:12590-12595.
Dimarcq, J.-L., D. Hoffmann, M. Meister, P. Bulet, R. Lanot, J.-M. Reichhart, and J. A. Hoffmann. 1994. Characterization and transcriptional profiles of a Drosophila gene encoding an insect defensin: a study in insect immunity. Eur. J. Biochem. 221:201-209.[Abstract]
Dushay, M. S., B. Åsling, B., and D. Hultmark. 1996. Origins of immunity: Relish, a compound Rel-like gene in the antibacterial defense of Drosophila. Proc. Natl. Acad. Sci. USA 93:10343-10347.
Ekengren, S., and D. Hultmark. 1999. Drosophila cecropin as an antifungal agent. Insect Biochem. Mol. Biol. 29:965-972.[CrossRef][ISI][Medline]
Ernst, J. D. 2000. Bacterial inhibition of phagocytosis. Cell. Microbiol. 2:379-386.[CrossRef][ISI][Medline]
Ernst, J. D. 2000. Hitchhiking under positive Darwinian selection. Genetics 155:1405-1413.
Fay, J. C., G. J. Wyckoff, and C.-I Wu. 2002. Testing the neutral theory of molecular evolution with genomic data from Drosophila. Nature 415:1024-1026.[CrossRef][ISI][Medline]
Hudson, R. R. 1987. Estimating the recombination parameter of a finite population model without selection. Genet. Res. 50:245-250.[ISI][Medline]
Hudson, R. R. 1990. Gene genealogies and the coalescent process. Pp. 144 in D. Futuyma and J. Antonovics, eds. Oxford surveys in evolutionary biology. Oxford University Press, Oxford.
Hudson, R. R. 2002. Generating samples under a Wright-Fisher neutral model of genetic variation. Bioinformatics 18:337-338.
Hudson, R. R., M. Kreitman, and M. Aguadé. 1987. A test of neutral molecular evolution based on nucleotide data. Genetics 116:153-159.
Hueck, C. J. 1998. Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol. Mol. Biol. Rev. 62:379-433.
Hughes, A. L., and M. Nei., Pattern of nucleotide substitution at major histocompatibility complex class I loci reveals overdominant selection. 1988. Nature 335:167-70.
Irving, P., L. Troxler, T. S. Heuer, M. Belvin, C. Kopczynski, J.-M. Reichhart, J. A. Hoffmann, and C. Hetru. 2001. A genome-wide analysis of immune responses in Drosophila. Proc. Natl. Acad. Sci USA 98:15119-15124.
Kelly, J. 1997. A test of neutrality based on interlocus associations. Genetics 146:1197-1206.
Kimura, M. 1983. The neutral theory of molecular evolution. Cambridge University Press, Cambridge.
Kirby, D. A., and W. Stephan. 1995. Haplotype test reveals departure from neutrality in a segment of the white gene of Drosophila melanogaster. Genetics 141:1483-90.
Kreitman, M. 1983. Nucleotide polymorphism at the alcohol dehydrogenase locus of Drosophila melanogaster. Nature 304:412-417.[ISI][Medline]
Kumar, S., K. Tamura, I. B. Jakobsen, and M. Nei. 2001. MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17:1244-1245.
Lazzaro, B. P., and A. G. Clark. 2001. Evidence for recurrent paralogous gene conversion and exceptional allelic divergence in the attacin genes of Drosophila melanogaster. Genetics 159:659-671.
Lee, J.-Y., A. Boman, S. Chuanxin, M. Andersson, H. Jörnvall, V. Mutt, and H. G. Boman. 1989. Antibacterial peptides from pig intestine: isolation of a mammalian cecropin. Proc. Natl. Acad. Sci. USA 86:9159-9162.[Abstract]
Lee, I. H., Y. Cho, and R. I. Lehrer. 1997. Styelins, broad-spectrum antimicrobial peptides from the solitary tunicate, Styela clava. Comp. Biochem. Physiol. 118B:515-521.[CrossRef]
Lehninger, A. L., D. L. Nelson, and M. M. Cox. 1993. Principles of biochemistry. Worth Publishers, New York.
Levashina, E. A., S. Ohresser, P. Bulet, J.-M. Reichhart, C. Hetru, and J. A. Hoffmann. 1995. Metchnikowin, a novel immune-inducible proline-rich peptide from Drosophila with antibacterial and antifungal properties. Eur. J. Biochem. 233:694-700.[Abstract]
McDonald, J. H., and M. Kreitman. 1991. Adaptive protein evolution at the Adh locus in Drosophila. Nature 351:652-654.[CrossRef][ISI][Medline]
Przeworski, M. 2002. The signature of positive selection at randomly chosen loci. Genetics 160:1179-1189.
Ramos-Onsins, S., M. Aguadé. 1998. Molecular evolution of the cecropin multigene family in Drosophila: functional genes vs. pseudogenes. Genetics 150:157-171.
Rozas J., and R. Rozas. 1999. DnaSP version 3: an integrated program for molecular population genetics and molecular evolution analysis. Bioinformatics. 15:174-175.
Rozas J., and R. Rozas. 1990. The immune response in Drosophila: pattern of cecropin expression and biological activity. EMBO J. 9:2969-2976.[Abstract]
Sokal, R. R., and F. J. Rohlf. 1995. Biometry, 3rd edition. W. H. Freeman and Company, New York.
Sugiyama, M., H. Kuniyoshi, and E. Kotani, et al. (14 co-authors). 1995. Characterization of a Bombyx mori cDNA encoding a novel member of the attacin family of insect antibacterial peptides. Insect Biochem. Mol. Biol. 25:385-392.[CrossRef][ISI][Medline]
Tajima, F., 1989 Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123:585-595.
Tryselius, Y., C. Samakovlis, D. A. Kimbrell, and D. Hultmark. 1992. CecC, a cecropin gene expressed during metamorphosis in Drosophila pupae. Eur. J. Biochem. 204:395-399.[Abstract]
Wayne, M. L., D. Contamine, and M. Kreitman. 1996. Molecular population genetics of Ref(2)P, a locus which confers viral resistance in Drosophila. Mol. Biol. Evol. 13:191-199.[Abstract]
Wicker, C., J-M. Reichhart, D. Hoffmann, D. Hultmark, C. Samakovlis, and J. A. Hoffmann. 1990. Insect immunity: characterization of a Drosophila cDNA encoding a novel member of the diptericin family of immune peptides. J. Biol. Chem. 265:22493-22498.
Wiehe T. H., and W. Stephan. 1993. Analysis of a genetic hitchhiking model, and its application to DNA polymorphism data from Drosophila melanogaster. Mol. Biol. Evol. 10:842-854.[Abstract]
Zhao, C., L. Liaw, I. H. Lee, and R. I. Lehrer. 1997. cDNA cloning of three cecropin-like antimicrobial peptides (styelins) from the tunicate, Styela clava. FEBS Lett. 412:144-148.[CrossRef][ISI][Medline]
Zhou, X., T. Nguyen, and D. A. Kimbrell. 1997. Identification and characterization of the cecropin antibacterial protein gene locus in Drosophila virilis. J. Mol. Evol. 44:272-281.[ISI][Medline]