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Genetics. Apr 2005; 169(4): 2023–2034.
PMCID: PMC1449580

Elevated Polymorphism and Divergence in the Class C Scavenger Receptors of Drosophila melanogaster and D. simulans


Scavenger receptor proteins are involved in the cellular internalization of a broad variety of foreign material, including pathogenic bacteria during phagocytosis. I find here that nonsynonymous divergence in three class C scavenger receptors (Sr-C's) between Drosophila melanogaster and D. simulans and between each of these species and D. yakuba is approximately four times the typical genome average. These genes also exhibit unusually high levels of segregating nonsynonymous polymorphism in D. melanogaster and D. simulans populations. A fourth Sr-C is comparatively conserved. McDonald-Kreitman tests reveal a significant excess of replacement fixations between D. melanogaster and D. simulans in the Sr-C's, but tests of polymorphic site frequency spectra do not support models of directional selection. It is possible that the molecular functions of SR-C proteins are sufficiently robust to allow exceptionally high amino acid substitution rates without compromising organismal fitness. Alternatively, SR-Cs may evolve under diversifying selection, perhaps as a result of pressure from pathogens. Interestingly, Sr-CIII and Sr-CIV are polymorphic for premature stop codons. Sr-CIV is also polymorphic for an in-frame 101-codon deletion and for the absence of one intron.

SCAVENGER receptors (SRs) compose a protein family defined by gross structural similarities and participation in cellular internalization of foreign compounds. In contrast to the extreme specificity exhibited by most receptor-ligand systems, individual scavenger receptors have high affinity for a broad array of polyanionic ligands such as bacteria, apoptotic cell debris, and modified lipopolyprotein (reviewed in Krieger et al. 1993). Drosophila melanogaster Sr-CI, the one functionally characterized class C scavenger receptor (SR-C), shares with mammalian class A scavenger receptors (SR-As) the ability to bind compounds such as acetylated low-density lipopolyprotein, intact gram-negative and gram-positive bacteria, and bacterial molecular components (Abrams et al. 1992; Krieger and Herz 1994; Pearson et al. 1995; Krieger 1997; Gough and Gordon 2000). SR-CI and SR-As require interaction with other, unknown proteins at the membrane surface to internalize bound targets (met et al. 2001; Underhill and Ozinsky 2002; Meister 2004). Sr-A mutant mice are susceptible to bacterial infection (Peiser et al. 2002) and Sr-CI-deficient D. melanogaster cells fail to phagocytose bacteria at wild-type levels (met et al. 2001). Naturally occurring polymorphism in D. melanogaster Sr-C's has been associated with phenotypic variation in the ability to suppress bacterial infection (Lazzaro et al. 2004). Due to the potential importance of Sr-C's in pathogen recognition and host defense, understanding the evolutionary pressures experienced by these genes is of interest.

Three “predicted” gene homologs of Sr-CI (CG4099) can be recovered from the Drosophila melanogaster complete genome sequence (Sr-CII, CG8856; Sr-CIII, CG31962; Sr-CIV, CG3212). Because Sr-CI has been most thoroughly functionally characterized of the four, it will be considered the prototypical Sr-C throughout this article. Domain structures of the gene products from all four Sr-C's are presented in Figure 1. The N terminus of the mature SR-CI protein is extracellular and is defined by two tandem complement-control protein (CCP) domains and one MAM domain. The CCP and MAM domains are sufficient for binding of bacteria (met et al. 2001). A short spacer separates the MAM domain from a somatomedin B domain with unknown function and a long threonine-rich domain that extends the extracellular portion of the protein away from the membrane surface. The C terminus of SR-CI is cytoplasmic and may be heavily phosphorylated (Pearson et al. 1995). SR-CII is identical in domain structure to SR-CI, but SR-CIII and SR-CIV are putatively extracellularly secreted and lack Thr-rich, transmembrane and cytoplasmic domains. SR-CIII additionally lacks the somatomedin B domain (Figure 1). The precise functions of SR-Cs II, III, and IV have not been elucidated, but given their homology to SR-CI and apparent contribution to antibacterial immunocompetence (Lazzaro et al. 2004), I consider that these proteins, like SR-CI, may be involved in the recognition of pathogens or pathogen-derived molecules.

Figure 1.
Schematic of the four Drosophila Sr-C proteins. Protein functional domains are demarcated after Pearson et al. (1995). Intron positions in the corresponding genes, indicated only for Sr-CI, are conserved across the four genes.

Patterns of molecular variation have been used to infer evolutionary forces acting on many classes of proteins involved in the recognition of pathogens and foreign compounds. These data do not, however, paint a uniformly consistent picture. The archetypal recognition structure has been the vertebrate major histocompatibility complex (MHC), involved in the presentation of antigens to the immune system. In particular, the antigen-binding cleft of the MHC is extremely polymorphic. This variability expands the range of efficiency of antigen presentation by the MHC and is maintained by overdominant, diversifying natural selection (Hughes and Nei 1988, 1999). Insects lack antibody-mediated immune responses, however, and are not known to have MHC-equivalent molecules. The best-characterized recognition proteins in invertebrates are pattern recognition receptors (PRRs) such as peptidoglycan recognition proteins (PGRPs) and gram-negative binding proteins (GNBPs). PRRs (also present in vertebrates) recognize microbial molecules such as peptidoglycan and β-glucan using functional domains that are highly conserved across distantly related species (e.g., Dziarski 2004). PRR genes harbor very little intraspecific polymorphism (Jiggins and Hurst 2003; Little et al. 2004; B. P. Lazzaro and A. G. Clark, unpublished observations). The prevailing hypothesis is that the molecules bound by PRRs cannot tolerate extensive structural alteration, preventing microbes from evolving evasion and placing little pressure for adaptation on host PRRs. The PRR-based recognition system is aided by the fact that the target epitopes are unambiguously microbial, obviating the need for the host to make fine distinctions between self and non-self.

The expectation with respect to SR-C diversity levels is unclear. On one hand, SR-C diversity may be unnecessary if, like GNBPs and PGRPs, SR-Cs are responsive to conserved, easy-to-recognize epitopes such that potential pathogens cannot evolve evasion. Preliminary surveys, however, have suggested that patterns of Sr-C molecular diversity in wild North American populations of D. melanogaster (B. P. Lazzaro and A. G. Clark, unpublished observations) and D. simulans (Schlenke and Begun 2003) are more complex than those of PGRPs and GNBPs. To more thoroughly characterize variability in Drosophila Sr-C's, I have sampled alleles of all four Sr-C genes from both North American and African populations of D. melanogaster and D. simulans. Diversity levels are measured across gene regions encoding putative functional domains, and statistical tests are employed to detect evidence of potential adaptive evolution. The collected data are interpreted in the context of the current understanding of variability in invertebrate immune response genes specifically and the D. simulans and D. melanogaster genomes in general.


Origin of the Drosophila lines studied:

Alleles were sampled from African and North American D. melanogaster and D. simulans populations. The North American D. melanogaster (n = 12) are chromosome 2 extracted lines derived from flies collected in Pennsylvania in 1998. These lines have previously been used to survey variation in genes encoding secreted antibacterial peptides (Lazzaro and Clark 2001, 2003). African D. melanogaster (n = 10) were collected in 1992 from the Sangwa wildlife refuge in Zimbabwe and have since been maintained as isofemale lines (Begun and Aquadro 1993). These lines were provided by C. F. Aquadro. North American D. simulans (n = 8) were collected in northern California in 1995 and have previously been used to survey variation at a large number of immunity-related and immunity-independent loci (Begun and Whitley 2000a; Schlenke and Begun 2003). African D. simulans (n = 9) were collected in Harare, Zimbabwe, in 1994 by associates of C. F. Aquadro and have since been maintained as isofemale lines (C. F. Aquadro, personal communication). The Drosophila yakuba strain sequenced was obtained from the Drosophila Species Stock Center in Tuscon, Arizona.

Generation and analysis of sequence data:

Sr-CIII and Sr-CI are tandemly arranged head-to-tail at D. melanogaster cytological position 24D. A total of 3794 bp of this locus were surveyed, including the entire coding sequence of each gene (Sr-CIII, 960 bp; Sr-CI, 1908 bp), 161 bp 5′ of the SR-CIII start codon, 95 bp 3′ of the SR-CI stop codon, an ~300-bp intergenic spacer between the two genes, the two introns of Sr-CIII (118 bp), and the three introns of Sr-CI (240 bp). The Sr-CII survey region (cytological position 48F) begins at the start codon, includes the entire coding sequence (1804 bp) and three introns (350 bp), and terminates 6 bp 3′ of the stop codon. The Sr-CIV survey region (cytological position 23F) begins 208 bp 5′ of the start codon, includes the 1221-bp coding region and both introns (137 bp), and terminates 65 bp 3′ of the stop codon.

DNA was extracted from pools of ~30 flies from each line by a standard phenol:chloroform extraction followed by ethanol precipitation. Primers to amplify and sequence each of the four Sr-C gene regions from D. melanogaster and D. simulans were designed using the D. melanogaster complete genome sequence (Adams et al. 2000). Primers for amplification and sequencing of D. yakuba were designed using data deposited by the D. simulans/D. yakuba genome sequencing consortium into the NCBI trace archive (http://www.ncbi.nlm.nih.gov/Traces). Primer sequences and PCR amplification conditions are available upon request. PCR products were directly sequenced using Beckman Coulter (Fullerton, CA) CEQ or ABI BigDye (Applied Biosystems, Foster City, CA) technologies under slight modification of the manufacturers' suggested protocols. All alleles were sequenced on both strands.

Initial sequencing of the African D. melanogaster lines revealed them to be highly heterozygous. For these lines only, DNA was re-extracted from a single fly representing each line and each locus was sequenced again. The presumption was that the individual flies were likely to be homozygous for one of the alleles segregating in each line and that choosing a random homozygous individual from a known heterozygous strain is analogous to randomly choosing a single allele from the population. The individual fly representing one line was heterozygous at Sr-CII, and the individuals from two other lines were heterozygous at the Sr-CIII,I locus. These lines were dropped from the analysis of the relevant loci (thus, n = 9 in Sr-CII and n = 8 in Sr-CIII,I). In Sr-CIV, one-half of the individual African D. melanogaster were heterozygous at Sr-CIV. For this locus, amplification products were cloned using a TOPO TA kit (Invitrogen, Carlsbad, CA) such that single alleles could be sequenced. At least two clones of each allele were sequenced, and then a single allele from each line was randomly chosen for inclusion in population genetic analyses. The same approach of cloning and sequencing was adopted for the D. yakuba strain, which also turned out to be heterozygous at Sr-C loci.

Partial coding regions of all four SR-C genes from the North American D. simulans lines considered here have been previously and independently sequenced by Schlenke and Begun (2003) and submitted to GenBank (accession nos. AY349846, AY349847, AY349848, AY349849, AY349850, AY349851, AY349852, AY349853, AY349854, AY349855, AY349856, AY349857, AY349858, AY349859, AY349860, AY349861, AY349878, AY349879, AY349880, AY349881, AY349882, AY349883, AY349884, AY349885, and AY349713, AY349714, AY349715, AY349716, AY349717, AY349718, AY349719, AY349720). The two sets of sequences are in nearly perfect agreement, although several ambiguous bases in the Schlenke and Begun sequences (scored as “N”) were resolved here. At the infrequent positions where there are disagreements between bases obtained here and those reported by Schlenke and Begun, the base sequence obtained here was used in analysis. D. yakuba sequences obtained by Schlenke and Begun were not included in the analyses in this article.

Data analysis:

Unless otherwise noted, molecular population genetic test statistics were calculated using DnaSP 3.51 and DnaSP 4.00 (Rozas and Rozas 1999; Rozas et al. 2003). Alleles with very large deletions were excluded from calculations of polymorphism and divergence to avoid eliminating too many informative sites. Calculation of statistics including all alleles and eliminating all sites with alignment gaps yielded comparable results (not shown). McDonald-Kreitman tests (McDonald and Kreitman 1991) were calculated using the 2 × 2 test of independence in DnaSP. McDonald-Kreitman tests were also run under a Poisson random field framework (Bustamante et al. 2002) on servers maintained by the Cornell Computational Biology Service Unit (Cornell CBSU) using default input parameters. K*ST (Hudson et al. 1992) and H (Fay and Wu 2000) were calculated using scripts written in C++. For calculation of H and for a subset of the McDonald-Kreitman tests, it is necessary to assign mutation events to either the D. melanogaster or the D. simulans lineages. These assignments were made assuming mutational parsimony and disallowing back mutation. Mutations that could not be parsimoniously placed on a single lineage (for instance, where D. yakuba and D. melanogaster are fixed for alternate states of a D. simulans polymorphism) were discarded from the relevant tests. Critical values of H were determined by simulation of 10,000 neutral genealogies after Hudson (1990) using Hudson's “mksamples” coalescence simulator (Hudson 2002), conditioning on the observed number of segregating sites and assuming no recombination. Critical values of K*ST were determined by randomly permuting subpopulation assignments of alleles and recalculating the test statistic 10,000 times.


Polymorphism and divergence:

Nonsynonymous divergence is substantially elevated in Sr-C's I, III, and IV between D. melanogaster and D. simulans and between D. melanogaster or D. simulans and D. yakuba. Synonymous nucleotide divergence is normal or slightly elevated across all species pairs in all genes (Table 1).

Per-site nonsynonymous (synonymous) Jukes-Cantor corrected nucleotide divergence across SR-C domains

The CCP and MAM domains of SR-CI have previously been shown to be sufficient for binding of bacteria (met et al. 2001). Nonsynonymous divergence in the sequence encoding these domains is approximately four times (CCP) and twice (MAM) the average level among the genomes of these three Drosophila species (Table 1; Takano 1998). The gene regions encoding the transmembrane domain and cytoplasmic tail of SR-CI show 2- to 3-fold excess nonsynonymous divergence. The Thr-rich domain, which is likely to be more mutable due to the repetitive nature of the sequence and less constrained by precise primary sequence than by tertiary structure, is encoded by sequence three to seven times more divergent at replacement positions than the genome averages. Most strikingly, the sequence of the functionally uncharacterized somatomedin B domain, which is perfectly conserved in length, has a nonsynonymous replacement rate equivalent to typical synonymous divergence rates in these species. The somatomedin B domain of Sr-CI shows a Jukes-Cantor corrected divergence of 8.5% in nonsynonymous positions between D. melanogaster and D. simulans and of 27.7% (26.4%) between D. melanogaster (D. simulans) and D. yakuba. Synonymous divergence between D. melanogaster (D. simulans) and D. yakuba is 33.3% (31.0%) in Sr-CI. By way of contrast, replacement divergence between D. melanogaster and D. simulans is typically on the order of 1.2% and between D. melanogaster (D. simulans) and D. yakuba is 2.5% (2.2%) (Takano 1998; see also Begun and Whitley 2000a, Begun 2002). Thus, nonsynonymous divergence in the somatomedin B domain of Sr-CI is increased ~10-fold relative to genome norms, with KaKs, a condition attributable to either a complete relaxation of purifying selection or strong and consistent diversifying selection (Yang and Bielawski 2000).

Sr-CIII, which consists of only the CCP and MAM domains, displays a rate of nonsynonymous divergence similar to that of the homologous regions of Sr-CI. Sr-IV, which has CCP, MAM, and somatomedin B domains, also has an overall divergence rate similar to that of Sr-CI, although fewer of the substitutions are in the somatomedin B sequence and more are in the MAM (Table 1).

Sr-CII is far less divergent between species than are the other Sr-C's and is generally in line with, although on the high end of, divergences observed in independent genes across these three species (Table 1; Takano 1998). The sequence encoding the Thr-rich domain of SR-CII displays nonsynonymous divergence equivalent to that of the corresponding sequence of Sr-CI, but this may reflect a relaxation of constraint on primary amino acid sequence of the Thr-rich domain (provided overall polarity is retained) or an increase in mutation rate due to the repetitive nature of the nucleotide sequence. In stark contrast to Sr-CI, the Sr-CII gene regions encoding CCP, MAM, and somatomedin B domains are highly conserved across species, actually diverging less than the genome average at nonsynonymous positions between D. melanogaster and D. simulans or between either species and D. yakuba (Table 1).

Paralleling the interspecific divergence data, intraspecific nonsynonymous polymorphism is also elevated in the Sr-C genes (Table 2). Replacement substitutions typically constitute 20–30% of the total number of polymorphic sites in D. melanogaster (Moriyama and Powell 1996; Andolfatto 2001; Fay et al. 2002; Mousset and Derome 2004) and 8–15% of polymorphic sites in D. simulans (Moriyama and Powell 1996; Begun and Whitley 2000a; Andolfatto 2001; Mousset and Derome 2004). The proportion of polymorphic sites in Sr-C's predicted to change amino acid sequence ranges from 37% (Sr-CII) to 63% (Sr-CIV) in D. melanogaster and 20% (Sr-CII) to 53% (Sr-CIV) in D. simulans (Table 3). The probability that a certain proportion of polymorphic sites are nonsynonymous can be treated as a binomial sampling process with Prob(success) equal to the mean proportion of polymorphisms that are nonsynonymous genome wide. Using this test, the excess of nonsynonymous relative to synonymous polymorphisms between individual Sr-C's and the relevant genome average is highly significant at Sr-C's I, III, and IV (P < 10−4), but not at Sr-CII (P = 0.06 in D. melanogaster; P = 0.04 in D. simulans).

Summary statistics describing polymorphism DrosophilaSr-C genes
McDonald-Kreitman comparisons of polymorphism and divergence

The observed increases in both nonsynonymous polymorphism and divergence could conceivably result either from positive selection acting to diversify Drosophila Sr-C's or from a relaxation of purifying selection on these genes relative to genome norms. An elevated mutation rate cannot explain the patterns observed in these genes because silent divergence (Table 1) and polymorphism (Table 2) are not substantially increased. The McDonald-Kreitman (MK) test can reveal heterogeneity between the ratio of synonymous to replacement polymorphisms within species compared to the ratio of synonymous to replacement fixations between species (McDonald and Kreitman 1991). Application of the MK test reveals a marginally significant excess of nonsynonymous fixations between D. melanogaster and D. simulans in Sr-CI (G = 4.446, P = 0.035) and nearly significant excesses of nonsynonymous fixations at Sr-CII and Sr-CIII (G = 3.198, P = 0.074 and G = 3.519, P = 0.061, respectively; Table 3). Exclusion of the sequence encoding the hypothetically unconstrained (and highly polymorphic) Thr-rich domains from analyses of Sr-CI and Sr-CII reveals a more substantial excess of nonsynonymous fixations in the remaining domains of these genes (in Sr-CI, G = 8.62 and P = 0.003; in Sr-CII, G = 7.82 and P = 0.005). The MK test statistic is not significant in Sr-CIV due to the extreme number of replacement polymorphisms (the 406 codon gene harbors 81 nonsynonymous polymorphisms across both D. melanogaster and D. simulans). When data from the four genes are pooled, the MK test indicates a highly significant excess of nonsynonymous fixations between species (G = 9.364, P = 0.002), consistent with positive selection driving the diversification of these genes.

Interpretation is slightly clouded, however, when sequence from D. yakuba is used to localize mutation events to either the D. melanogaster or the D. simulans lineage. When mutations are polarized in this way (Table 3), D. simulans shows a significant excess of nonsynonymous fixations at Sr-CI (G = 5.751, P = 0.016), a nearly significant excess in Sr-CIV (G = 3.163, P = 0.057), and a significant excess across all four genes combined (G = 5.825, P = 0.016). The effect is not seen in Sr-CII and Sr-CIII (G = 0.236, P = 0.627 and G = 0.259, P = 0.611, respectively). Again, exclusion of the sequence encoding the Thr-rich domain results in a more profound pattern in Sr-CI (G = 11.27, P = 0.0008). No significant MK tests are observed on the D. melanogaster lineage due to the much higher proportional level of nonsynonymous polymorphism in that species (Table 3), although Sr-CIII showed a tendency toward excess nonsynonymous fixation (G = 2.983, P = 0.084). When substitutions along the D. melanogaster lineage in all four genes are considered jointly, G = 1.928 (P = 0.165).

A similar finding of significant MK tests between D. melanogaster and D. simulans and along the D. simulans lineage, but not along the D. melanogaster lineage, was previously observed in the immune-inducive transcription factor Relish (Begun and Whitley 2000b). In Relish, failure to reject homogeneity resulted from a reduction in synonymous polymorphism on the D. melanogaster lineage, such that levels of synonymous and replacement heterozygosity were comparable. Because the number of fixed replacements was elevated on both lineages and similar between species, Begun and Whitley (2000b) concluded that positive selection probably acts on Relish in both species. As in Relish, replacement fixations in Sr-C's are elevated on both the D. melanogaster and the D. simulans lineages relative to genome norms.

To test whether amino acid replacements in SR-Cs may be adaptive, the average estimate of γ = 4Nes was determined for nonsynonymous substitutions in each of the four Sr-C genes under a Poisson random field framework (Bustamante et al. 2002). γ was estimated at 1.28 (SE = 0.65) in Sr-CI and 0.73 (SE = 0.48) in Sr-CIII, indicating that the average replacement fixation is moderately selectively favored. These values are typical for Drosophila genes (Bustamante et al. 2002). In Sr-CIV, γ was estimated to be −0.07 (SE = 0.33) suggesting that replacement substitutions in this gene are effectively neutral, an observation unusual for functional Drosophila genes (Bustamante et al. 2002). Nonsynonymous fixations in Sr-CII seem to have been more strongly favored, with γ estimated at 3.27 (SE = 0.99). Only 4 of 34 Drosophila genes examined by Bustamante et al. (2002) have estimated γ as large as or larger than that estimated from Sr-CII.

Analyses of site frequency spectra, population structure, and linkage disequilibrium:

Patterns in the distribution of allele frequencies at polymorphic sites are frequently used to infer departure from null models of selective neutrality. Two such tests are Tajima's D (Tajima 1989) and Fay and Wu's H (Fay and Wu 2000). Negative values of D result from an excessive proportion of polymorphic sites at which the rarer allele is at very low frequency in the population and may be attributed to mutational recovery after a strong directional selective event. Positive values of D occur when a high proportion of the polymorphic sites have both states at intermediate frequencies, as under some scenarios of balancing selection. H is analogous to D, except that it tests for an excess of sites at which the derived state is common, which can also result from positive directional selection. Application of the H test to the SR-C data yielded no significant results in any of the four genes in either species (data not shown). Similarly, no significantly negative values of D were observed (Table 4). There is thus no indication from the site frequency data that these genes have experienced recent directional selection. In two cases D is significantly positive (Table 4). In North American D. melanogaster SR-CII, D = +1.87 (P = 0.017), although this value is not statistically significant after correction for multiple tests. North American D. simulans have D = +2.08 (P = 0.003) in the Sr-CIII,I cluster, a value that is significantly positive at α < 0.05 even after Bonferroni correction.

Summary statistics describing DrosophilaSr-C gene regions

In none of the loci is there a significant difference between the frequency spectrum of synonymous and nonsynonymous polymorphisms. Values of D are slightly smaller for nonsynonymous substitutions than for synonymous polymorphisms in 3 of the 16 comparisons across the four genes and populations (the exceptions being North American D. melanogaster Sr-CI and Sr-CII and African D. melanogaster Sr-CIV). This pattern is significantly unexpected (P = 0.011) if comparisons are considered Bernoulli trials where either synonymous or nonsynonymous polymorphisms are equally likely to exhibit smaller D. The significant tendency for nonsynonymous polymorphisms to segregate at lower population frequencies than synonymous polymorphisms is consistent with a general action of purifying selection on amino acid mutation.

The North American and African populations of both species are highly significantly differentiated at all loci as measured by K*ST (Table 5; Hudson et al. 1992). In genes encoding membrane-bound SR-Cs, variability in North American populations is a subset of the variation present in African populations, with most mutations shared across populations or private to African samples. Genome-wide genetic differentiation of this sort has previously been observed between African and North American Drosophila populations in antibacterial peptide genes (Clark and Wang 1997) and several immunity-independent loci (e.g., Andolfatto 2001; Caracristi and Schlötterer 2003). The pattern is generally attributed to Africa being the ancestral home of the D. melanogaster and D. simulans species, with subsequent founding events leading to the colonization of North America (David and Capy 1988; Hamblin and Veuille 1999; Andolfatto 2001; Wall et al. 2002). The genes encoding secreted SR-Cs, Sr-CIII and Sr-CIV, are unusual in that up to 30% of the segregating polymorphisms are private to the North American samples (not shown). This is unexpected under a simple founding model and may be consistent with adaptation to a new environment.

Genetic differentiation between African and North American subpopulations

Both D. melanogaster and D. simulans also exhibit increased linkage disequilibrium in North American populations relative to African. In D. melanogaster, per-site estimates of 4Nc (Hudson 1987) are approximately four- and sixfold reduced in North America relative to Africa in the Sr--CIII,I cluster and Sr-CII, respectively (Table 4). Increased linkage disequilibrium on this order has previously been observed in non-African relative to African populations in other, functionally unrelated, loci around the genome (e.g., Andolfatto and Przeworski 2000; Andolfatto and Wall 2003). There is no apparent reduction in 4Nc at D. melanogaster Sr-CIV. In North American D. simulans, however, the situation is extreme and highly unusual. All of the segregating sites in all four genes (88 in the Sr-CIII,I cluster, 89 in Sr-CII, and 46 in Sr-CI) are blocked into only two or three major haplotypes at each locus. With such strong linkage disequilibrium, estimated 4Nc = 0. Such a complete arrangement of so-called “yin-yang” haplotypes involving a large number of sites is unheard of in either D. melanogaster or D. simulans (Begun and Whitley 2000a; Zhang et al. 2003; but see Rozas et al. 2001; Quesada et al. 2003). This observation is explored in more detail in the accompanying article by Schlenke and Begun (2005)(this issue).

Large deletions, premature termination, and evolutionary modification of start and stop codons:

Perhaps the most striking aspect of the polymorphism observed in the Drosophila Sr-C genes is the presence of large deletions segregating in Sr-CIV (Figure 2). One of these is an in-frame deletion eliminating 101 codons, including those encoding 50 amino acids of the MAM domain and 26 amino acids of the somatomedin B domain. This deletion is present in 3 of the 10 African D. melanogaster alleles considered here, but is absent from the North American D. melanogaster alleles sampled, and was not detected in a previous screen of 101 North American D. melanogaster alleles of Sr-CIV (Lazzaro et al. 2004). A second in-frame D. melanogaster deletion is an imprecise excision of the second Sr-CIV intron. This deletion begins seven bases into the intron and extends one base into the third exon, resulting in a net insertion of 2 amino acids into the MAM domain. Three of the 10 African D. melanogaster lines and 2 of the 12 North American D. melanogaster alleles carry the deletion, although genotyping data from the larger North American sample places its frequency at 32.7% (Lazzaro et al. 2004).

Figure 2.
Positions and population frequencies of polymorphic large deletions and premature stop codons in Sr-CIV. Asterisks indicate premature stop codons, open brackets mark the boundaries of deletions. Although the intron 2 absence deletion is present in only ...

Three frameshift deletions and two point mutations result in premature stop codons in Sr-CIV (Figure 2). Two of the deletions are found in the North American D. melanogaster sample, and one is found in the African D. simulans; all are singletons. The D. simulans deletion eliminates 169 bases of exon 2, resulting in a stop codon after 90 codons. One of the D. melanogaster deletions eliminates most of the first intron and 16 bases of the second exon, resulting in a premature termination after 85 codons. The other D. melanogaster stop is 4 bp in length and terminates the predicted protein after 252 amino acids. Three additional North American D. melanogaster alleles carry premature stops caused by point mutations, one harboring a unique stop 30 codons into the protein and the other two sharing a termination codon after 385 codons. The predicted wild-type protein length is 406 amino acids, so the latter pair of alleles could potentially retain functionality. On the other hand, the preponderance of large deletions and segregating premature stop codons raises the possibility that Sr-CIV may not be a functional gene at all, but instead may be a young pseudogene. This possibility is further examined in discussion.

Polymorphic premature stop codons are not unique to Sr-CIV among the Drosophila Sr-C genes. Two D. melanogaster alleles of Sr-CIII, encoding the other putatively secreted SR-C, also harbor singleton point mutations resulting in truncation of the predicted protein sequence. One of these is a North American allele and ends the open reading frame after 10 codons. The other is an African allele that terminates after 22 codons. Interestingly, the Sr-CIII start codon varies among D. melanogaster, D. simulans, and D. yakuba. The predicted D. melanogaster protein sequence begins with the three amino acids Met-Ala-Met. It is not experimentally known which of the two methionine codons is actually the primary start in D. melanogaster, but the first methionine is absent from D. yakuba and the second methionine is mutated to leucine in D. simulans. Without sequence from an outgroup species, it is not possible to determine which Sr-CIII start codon is ancestral.

One African D. melanogaster allele of Sr-CII carries a four-base deletion in the last two codons of the gene. A stop codon in the new reading frame terminates translation, altering the C terminus of the protein from DL* to ERG*. There are no premature stop codons in Sr-CII, and there is no variability in start or stop in Sr-CI.


The class C scavenger receptors compose a four-member gene family in D. melanogaster, D. simulans, and D. yakuba (Figure 1). D. melanogaster SR-CI directly binds bacteria to facilitate phagocytosis (met et al. 2001) and is involved in endocytosis of lipopolyprotein (Abrams et al. 1992; Pearson et al. 1995). Detailed functional characterization of the other three genes is lacking, although naturally occurring polymorphism in all four D. melanogaster Sr-C genes has been implicated as contributing to phenotypic variation in the suppression of bacterial infection (Lazzaro et al. 2004). It seems reasonable to hypothesize that these genes may be important for the recognition of bacteria during an immune response, raising the question of what evolutionary pressures they face. Population genetic analysis of this Sr-C gene family in two populations each of D. melanogaster and D. simulans reveals them to be on different evolutionary trajectories.


Sr-CI shows rapid nonsynonymous divergence in the gene regions encoding the CCP, MAM, somatomedin B, and transmembrane/cytoplasmic domains (Table 1), all of which are conserved in length and unambiguously alignable across species. In particular, the rate of nonsynonymous divergence in the sequence encoding the Sr-CI somatomedin B domain is approximately equivalent to the rate of synonymous divergence (KaKs), a condition attributable to either positively selected diversification or a complete relaxation of functional constraint. The gene encoding the putatively secreted SR-CIII, which consists of only CCP and MAM domains, exhibits divergence rates similar to those of the homologous regions in Sr-CI.

McDonald-Kreitman tests reveal a significant excess of nonsynonymous fixations in Sr-CI between D. melanogaster and D. simulans (G = 4.446, P = 0.035; G = 8.62, P = 0.003 excluding the Thr-rich domain) and along the D. simulans lineage (G = 5.751, P = 0.016; G = 11.27, P = 0.0008 excluding the Thr-rich domain). The excess of replacement fixations in Sr-CIII is nearly significant between species (G = 2.983, P = 0.084). When considered jointly, these two genes show a highly significant excess of nonsynonymous fixations between D. melanogaster and D. simulans (G = 7.167, P = 0.007) consistent with adaptively driven divergence in these two genes, particularly in Sr-CI. Nonsynonymous fixations are, on average, estimated to be selectively favored in Sr-CI and Sr-CIII (γ = 1.27 in Sr-CI and 0.73 in Sr-CIII), although these values are unexceptional for Drosophila (Bustamante et al. 2002).

If the elevated nonsynonymous divergence observed in these genes were driven by pathogen diversity, intuition would suggest that substitutions would be concentrated in regions of the protein involved in binding. Contrary to that intuition, high amino acid divergence is also suggested in the SR-CI somatomedin B and transmembrane domains, neither of which is known to have direct interactions with bacteria. Interestingly, a similar substitution pattern is observed in mouse SR-A, where a small number of strain-specific nucleotide differences result in a high proportion of amino acid polymorphisms localized in regions of the protein flanking the interior and exterior of the membrane surface (Daugherty et al. 2000; Fortin et al. 2000). Although SR-CI and SR-A share ligand affinity and may have similar three-dimensional structures, they are unrelated in primary amino acid sequence and their functional similarities have arisen through evolutionary convergence. Amino-carboxy orientation is reversed in SR-A relative to SR-CI and the domain structure is completely distinct (Peiser et al. 2002). Given that SR-As and SR-Cs are structural analogs, not homologs, the convergence in substitution patterns may indicate similarity in evolutionary pressures experienced by those genes.

Why might the highest rates of substitution in both SR-CI and SR-A be observed in regions of the protein presumably involved not in pathogen binding but in protein-protein interactions? It is well known that pathogenic bacteria can actively inhibit host immune responses through a variety of mechanisms (e.g., Lindmark et al. 2001; Fauvarque et al. 2002). One could speculate that pathogens might also seek to evade engulfment by disrupting interactions between scavenger receptors and other proteins required for phagocytotic internalization, and then SR domains outside those responsible for direct bacterial binding may experience pressure driving evolutionary diversification. Begun and Whitley (2000b) put forward a similar hypothesis to explain the rapid evolution of the immune-related transcription factor Relish. The sequence encoding the Relish domain that is cleaved to activate the transcription factor shows extraordinarily high levels of nonsynonymous substitution, which Begun and Whitley (2000b) proposed is driven by the secretion into host cells of pathogen repressor molecules designed to prevent Relish activation. This hypothesis was later bolstered by the observation that Dredd, a caspase involved in the activation of Relish, shows correlated rapid evolution (Schlenke and Begun 2003).


In contrast to Sr-C's I and III, Sr-CII shows a high degree of conservation among D. melanogaster, D. simulans, and D. yakuba (Table 1). The gene regions encoding the MAM and CCP domains show higher than expected conservation in comparisons of D. melanogaster/D. simulans to D. yakuba, although divergence is slightly elevated in sequences encoding the somatomedin B and transmembrane domains (Table 1). The gene region encoding the threonine-rich domain exhibits high levels of replacement polymorphism and divergence in Sr-CII, likely due to the repetitive nature and presumed low functional constraint on the primary sequence of this domain, which is also variable in length between D. yakuba and D. melanogaster/D. simulans. It is conservative to imagine that many amino acid substitutions in this domain are functionally neutral. A McDonald-Kreitman test reveals a nearly significant excess of replacement fixations between D. melanogaster and D. simulans (G = 3.198, P = 0.074; G = 7.82, P = 0.005 when the Thr-rich domain is excluded), consistent with the action of positive selection on this gene, although it is clear that Sr-CII does not evolve under strong diversifying selection. The mean γ = Nes estimated for nonsynonymous substitutions in Sr-CII is 3.27, indicating adaptive favorability of the nonsynonymous fixations observed in this locus. This value is in the top 10% of estimates collected from a panel of unrelated Drosophila genes (Bustamante et al. 2002).

Published data suggest that Sr-CII is expressed only in early embryos (met et al. 2001), although low-level tissue-specific expression remains a possibility. This observation, coupled with the molecular evolutionary data, suggests that SR-CII may be functionally distinct from the other SR-Cs. As of June 2004, a BLAST search of the unassembled D. pseudoobscura genome sequence database at the Baylor College of Medicine (http://www.hgsc.bcm.tmc.edu/blast/?organism=Dpseudoobscura) yielded a high-quality match only to the comparatively conserved Sr-CII, but no clear matches to Sr-CI, Sr-CIII, or Sr-CIV (B. P. Lazzaro, unpublished observations).


The data from Sr-CIV are perplexing. Five of 12 North American D. melanogaster alleles sampled carry premature stop codons, and 3 of 10 African D. melanogaster alleles carry an in-frame 101-codon deletion. One of the 18 D. simulans Sr-CIV alleles sampled carries a premature stop codon. (Premature stops are not exclusive to Sr-CIV, as two alleles of Sr-CIII, the other secreted SR-C, are also predicted to terminate early in the protein.) The prevalence of stop codons and an apparently disruptive deletion in Sr-CIV raises the possibility that Sr-CIV may be a young pseudogene, at least in D. melanogaster, resulting in an absolute relaxation of constraint. If Sr-CIV is a pseudogene, though, it is an extremely young one, and may even be polymorphic for activity in D. melanogaster. Divergence among species is not substantially higher in Sr-CIV than in the other Sr-C genes, and there is no evidence for an acceleration of nonsynonymous divergence on the D. melanogaster lineage. Synonymous polymorphism and divergence far exceed nonsynonymous polymorphism and divergence in Sr-CIV, suggesting at least an historical functional constraint on mutations. McDonald-Kreitman tests yield no evidence of adaptive diversification in Sr-CIV (Table 3) and the estimate of γ (=Nes) = −0.07 on nonsynonymous fixations is completely consistent with neutrality. Ka/Ks is 0.5 between D. melanogaster and D. simulans and ~0.4 between either of these species and D. yakuba. On balance, the data suggest that functional constraint on Sr-CIV is greatly relaxed, although probably recently so. The additional presence of null alleles in Sr-CIII suggests that secreted SR-Cs may be dispensable with minimal effect on organismal fitness. Very young immune-related pseudogenes have previously been observed in Cecropin gene family in the D. melanogaster species subgroup, where two apparent pseudogenes in D. melanogaster continue to be transcribed despite exhibiting polymorphism for nonsense mutations (Ramos-Onsins and Aguadé 1998). A naturally occurring null allele has also been recovered in the gene encoding the antibacterial peptide Attacin A (Lazzaro and Clark 2001).

Sr-CIV is also exceptional in being polymorphic for the presence/absence of an intron, with the intron-absent state segregating at ~40% frequency in North American and African D. melanogaster. To date, only one other intermediate-frequency intron presence-absence polymorphism has been described from any eukaryote, in the jingwei gene of D. teissieri (Llopart et al. 2002). As in jingwei, the Sr-CIV polymorphism derives from an imprecise genomic deletion that eliminates the intron but retains reading frame.

Linkage disequilibrium in North American D. simulans:

The North American D. simulans lines are clear outliers with respect to the other lines in terms of linkage disequilibrium. The 88 polymorphic sites in the Sr-CIII,I locus segregate in only two major haplotypes (with two additional haplotypes separated by three mutations), the 89 sites in Sr-CII form two haplotypes, and the 46 sites in Sr-CIV form three major haplotypes (with a fourth distinguished by two mutations). The failure to find such strong haplotype structure outside the Sr-C's and two other immunity-related genes (Schlenke and Begun 2005, this issue) even though 56 additional genes have been surveyed in these same lines (Begun and Whitley 2000a; Schlenke and Begun 2003) largely precludes any hypotheses based on demography or admixture. Chromosomal inversions are an unlikely and unparsimonious explanation, since the same phenomenon is observed in Sr-C's on both arms of chromosome 2 and disequilibrium is not absolute between the physically proximal Sr-CIII,I locus at (D. melanogaster) cytological position 24D and Sr-CIV at 23F. Sr-CII has probably been affected by a strong selective sweep at a genetically linked locus conferring insecticide resistance (Schlenke and Begun 2004), but such strong linked sweeps are expected to be rare. Furthermore, Schlenke and Begun report that disequilibrium decays in regions flanking the Sr-C loci and that the haplotype structure observed in the D. simulans lines described here is not present in other populations sampled from North America (Schlenke and Begun 2005, this issue). Significant, although less severe, haplotype structure previously observed in European D. simulans has been attributed to the actions of positive selection (Rozas et al. 2001; Quesada et al. 2003).


Drosophila class C scavenger receptors are a multigene family exhibiting different evolutionary trajectories. Sr-C's I, III, and IV are rapidly evolving. In the case of Sr-CI and, possibly, in Sr-CIII, rapid evolution seems to be driven by positive selection in both D. melanogaster and D. simulans. D. melanogaster Sr-CIV, however, seems to be evolving under a lack of functional constraint and may be a young (or even polymorphic) pseudogene. In contrast to the other Sr-C's, Sr-CII is evolutionarily conserved, although it too displays indications of adaptive evolution. It will be interesting to couple future functional studies of these genes with the molecular evolutionary observations. It will be of particular interest to determine the degree to which the extensive nonsynonymous polymorphism exhibited by Drosophila Sr-C's influences protein function and whether there is any detriment to animals carrying apparent null mutations in the secreted Sr-C's. Notably, Sr-CI may be the first Drosophila pathogen recognition gene characterized as evolving under adaptive diversification.


I extend special thanks to Todd Schlenke for open discussion of unpublished results and sharing of D. simulans lines. I also thank Chip Aquadro for flies and Brian Bettencourt for assistance in searching the unassembled D. pseudoobscura genome sequence for SR-C homologs. Early stages of this work were supported by National Institutes of Health grant AI46402 to A. G. Clark. This research was facilitated by prompt public release of data by the D. simulans and D. yakuba genome sequencing consortium. Finally, I acknowledge the Institute for Drosophila Immunomics in Ithaca, New York.


Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. AY865019, AY865135.


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