Anopheles gambiae individuals were collected from West Africa ('BK': Burkina Faso, Koubri village, 12°11'54 N; 1°23'43W) and East Africa ('KY': Kenya, Mbita, Suba District). Anopheles arabiensis individuals were also collected from West Africa ('BK', in the same collections as An. gambiae, above) and East Africa ('TZ', Tanzania, Ifakara). All An. gambiae and An. arabiensis used in this study were provided by H. M. Ferguson (University of Glasgow, UK). Anopheles melas individuals were collected from Coastal Ghana (Ghana, Essiama, 4°57.4 N; 2°24.1W) by N. Tuno (Institute of Tropical Medicine, Nagasaki University, Japan). Anopheles merus and Anopheles quadriannulatus species A (hereafter An. quadriannulatus) were both obtained from laboratory colonies, maintained by the Medical Research Council of South Africa (provided by R. Maharaj; MRC, Durban, South Africa) and the University of Wageningen (Strain 'Sangqua', Zimbabwe, provided by W. Takken), respectively.

The species identity of all gambiae complex members was verified by diagnostic PCR 35, and the M and S molecular forms of An. gambiae were distinguished by PCR-RFLP [e.g. 36]. As expected from their known geographic distributions 37, all KY An. gambiae individuals were S form, and all but two of the BK An. gambiae sample were M-form. All An. gambiae individuals were also surveyed for 2La/+ chromosomal inversion status, using the PCR assay of White et al 38, derived from the sequenced breakpoints 39. As reported previously, in addition to the expected diagnostic 207 bp and 492 bp fragment lengths, these primers were found to amplify fragments of lengths ca. 687 bp, 672 bp, 760 bp and 1020 bp in some individuals 32. Direct sequencing of these fragments from a subset of individuals suggest they are insertion/deletion derivatives of expected assay products 3240, and within the polymorphic KY population the 2La/2L+^a amplification fragments were in Hardy-Weinberg equilibrium (51 individuals, χ² = 0.44, 1df., p = 0.51), allowing us to tentatively assign 2La/2L+^a inversion status to all individuals 32.

Thirty-two loci were selected for sequencing and analysis, including 16 of the 18 serpins currently identified in the An. gambiae genome (M. Kanost and K. Michel, pers. comm.), and 16 other protein-coding loci chosen to match the genomic position of the serpins without regard to function. SRPN19 (a non-inhibitory serpin with 1:1:1 orthologs in Drosophila melanogaster, Aedes aegypti and An. gambiae) and SRPN13, which does not appear in the current An. gambiae assembly, were not sequenced. The total sequenced length was ~19 Kbp of coding sequence per individual (i.e. approximately 600 bp from each locus; range 240 bp–800 bp). Not all loci were sequenced from the same individuals within populations, and not all loci were amplified from An. melas. A full summary of gene names, locations, and classification is presented in Table 1.

The 'control' loci should represent an unbiased sample of Anopheles genes, to which serpins can be compared. Because these control genes are position-matched, each lying ~90 Kbp (range 40–125 Kbp) from a 'partner' serpin, they should control for the effects of large-scale position-based variation in recombination and mutation rates. Note that improvements to the An. gambiae annotation have subsequently identified control locus 5 (previously annotated as ENSANGG000008091) as deriving from a retrotransposon (AgamP3.4, July, 2007).

Genomic DNA was extracted from single mosquitoes using DNeasy kits (QIAgen). PCR primers were designed from the published An. gambiae genome sequence 41, and the final primer sequences selected after trouble-shooting (sequences are given in Additional file 1). Only one PCR amplicon was used per locus, thus sequences do not represent entire genes. Following PCR, unincorporated primers and dNTPs were removed using exonuclease I (New England BioLabs) and shrimp alkaline phosphatase (Amersham). PCR products were sequenced in both directions using BigDye™ reagents (v3.1, Applied BioSystems) and an ABI capillary sequencer. In some amplicons, indel polymorphism required the use of additional sequencing primers. The sequence chromatograms were assembled using SeqManII (DNAstar Inc., Madison USA) then inspected by eye to confirm the validity of all differences within and between species and all heterozygous base-calls. The heterozygous sequence from each diploid individual was decomposed into two pseudohaplotypes for analysis using PHASE 4243. However, the presented analyses should be highly robust to any errors in phase assignment, as only explicitly tree-based results, such as Hudson's nearest neighbour statistic (Snn), are affected by allelic phase. All unphased sequences have been submitted to GenBank as population sets, using ambiguity codes to indicate heterozygous sites. Sequence accession numbers span the range GQ146469–GQ148534.

The number of synonymous and non-synonymous polymorphisms and substitutions, and the average pairwise genetic diversity (π) were calculated using DnaSP [version 4.50.3, ref 44]. Diversity was calculated separately for synonymous (π_s) and non-synonymous (π_a) sites, and used a Jukes-Cantor correction for multiple substitutions, as implemented in DnaSP. Departures from the allele frequency spectrum expected under the standard neutral model were quantified using Tajima's D statistic 45, also calculated using DnaSP. Tajima's D (which measures departures from the expected allele frequency distribution under a standard neutral model) was calculated using synonymous sites only, and was calculated separately for both populations of An. gambiae and An. arabiensis (but not for An. melas, where sample sizes were too small to give meaningful results). The significance of departures from the expected allele frequency distribution were tested using 10,000 rounds of coalescent simulation (as implemented in DnaSP) conditional on the number of segregating sites and conservatively assuming no recombination within loci.

Genetic differentiation between populations was quantified in DnaSP using Hudson's K_ST statistic [equations 7 to 9 in reference 46] which is calculated from the average number of pairwise differences between sequences taken within populations and between all populations, and is identical to Nei's γ_ST 47 except for the population-size weighting scheme [see 46]. Significant departures from zero population differentiation were inferred by permuting sequences between populations to create a null distribution of K_ST values. All non-parametric statistical tests on diversity and differentiation were performed using the R statistical language (R Development Core Team, 2008 http://www.R-project.org). Although non-parametric tests (Spearman's rank correlation, paired Wilcoxon tests) are presented below, except where noted explicitly, parametric equivalents (Pearson's correlation, paired t-tests) gave qualitatively identical results.

If it is assumed that synonymous mutations are effectively neutral, and that the fixation or loss of selected amino-acid variants is so rapid that the vast majority of non-synonymous polymorphisms are also effectively neutral, then the relative numbers of polymorphisms (P, within species) and fixed differences (D, between species) at synonymous and non-synonymous sites can be used to identify the action of selection [see 7 for an introduction]. This forms the basis of the McDonald-Kreitman test [MK test, 29], which seeks to detect a departure from independence in a simple 2 × 2 contingency table of polymorphisms (P_N and P_S) and fixed differences (D_N and D_S). For a single gene, the departure from neutrality can easily be quantified by summary statistic such as the neutrality index (N.I.= (P_N/P_S)/(D_N/D_S) 48), or by the estimated proportion of adaptive substitutions (α = 1 - (D_SP_N)/(D_NP_S) = 1 - N.I., 49). This approach can be extended to multiple genes using D_S, P_N, D_N and P_S averaged across genes 49, or using a more sophisticated maximum-likelihood estimator of α, such as that of Bierne and Eyre-Walker 50.

Here, an extension of the maximum-likelihood method of Welch 30 was used. This method is very closely related to that of Bierne and Eyre-Walker 50, but additionally allows for the possibility that some apparent fixed differences may actually be polymorphisms that only appear fixed due to small sample size 51. The method was extended to include polymorphism values from two species simultaneously e.g. 2951. Using this approach, models were fitted in which expected neutral divergence, λ = μt, took a single value at all loci; expected neutral diversity, θ = 4N_eμ, was also shared between all loci, but free to vary between species; and selective constraint, f, was free to vary between loci [see 30 for other details of the model]. Three nested models were fitted, in which (1) α was constrained to zero at all loci, i.e. no adaptive evolution, (2) a single α was shared by all genes, and (3) α was free to differ between serpins and 'control' genes. In this way, it was possible to test both for evidence of adaptive evolution, and whether serpins have a different rate of adaptive evolution to other genes. Model fit was tested using both likelihood ratio tests and Akaike weighting (derived from the Akaike information criterion) 52. Confidence intervals on α were obtained by adjusting α away from its maximum likelihood value, and allowing the other parameters to take their maximum likelihood value, conditional on that α, until log likelihood decreased by 2 units 50. C code to fit these models is available on request from the authors, or from 53.

Across all 32 loci, average pairwise genetic diversity at synonymous sites (π_s) was highest in An. gambiae (π_s = 2.82%, 95% bootstrap interval 2.25–3.41%), lowest in An. melas (π_s = 0.86%, 0.53–1.19%), and intermediate in An. arabiensis (π_s = 1.97%, [1.50, 2.46]; Figure 1a). The difference in π_s between An. gambiae and An. arabiensis was highly significant (Paired Wilcox test using 32 loci, V = 444, p = 0.0004), and π_s correlated strongly between these species (Spearman's rho = 0.72, S = 1504, p < 6 × 10^-6, Additional file 2). Diversity in An. gambiae and An. arabiensis did not correlate with diversity in An. melas (p > 0.5 in both cases, 26 loci). For a full summary of synonymous diversity, and all other summary stats that follow, see Additional file 3.

For An. gambiae, genetic diversity was slightly higher in West Africa (π_s = 2.74%; BK M-form) than in East Africa (π_s = 2.17%; KY S-form), and although the effect was small, it was statistically significant (Paired Wilcox V = 380, p = 0.03, Figure 1a). Diversity was also highly correlated between East- and West-African populations (Spearman's rho = 0.72, S = 1520, p = 3 × 10^-6). Although only two S-form individuals were sampled from West Africa, they displayed higher diversity than the either of the other two An. gambiae populations (BK S-form; π_s = 2.98%). For An. arabiensis, diversity correlated even more strongly between East and West Africa (rho = 0.87, S = 729, p < 2 × 10^-10) and did not differ significantly between the populations (π_s = 1.96% in BK vs. π_s = 1.79% in TZ, Paired Wilcoxon test V = 316, p = 0.19).

Synonymous site diversity did not differ significantly between serpins and other genes in either An. gambiae (3.10% vs. 2.53%, Paired Wilcoxon V = 48, p = 0.32, Figure 1a) or An. arabiensis (2.00% vs. 1.93%, Paired Wilcox V = 60, p = 0.71). Although position-matched, no correlation in diversity could be detected between serpins and their corresponding control genes (p > 0.1 in both cases), suggesting that the effect of genomic location on neutral diversity was relatively weak.

Non-synonymous diversity (π_a) was very similar between An. gambiae (π_a = 0.22%; 95% bootstrap interval 0.13–0.31%) and An. arabiensis (π_a = 0.18%; bootstrap interval: 0.12–0.24%, Figure 1b), and did not differ significantly between the species (Paired Wilcoxon test V = 285, p = 0.29). Although π_a did not correlate significantly with π_s in either An. gambiae or An. arabiensis (correlation coefficients were 0.17 and 0.07 respectively, p > 0.3 in both cases), there was a strong correlation in π_a between the two species (rho = 0.81, S = 1037, p = 2 × 10^-8, Additional file 2). East and West African populations did not differ significantly in π_a for either An. gambiae or An. arabiensis (p > 0.1 in both cases), but π_a did correlate very highly between East and West African populations (rho = 0.80 p = 6. × 10^-8, and rho = 0.88, p = 2 × 10^-11, respectively). Interestingly, π_a was higher for serpins than for other genes in both An. gambiae and An. arabiensis (π_a = 0.30% vs. 0.13%, and π_a = 0.25% vs. 0.11%), although statistical significance was marginal for An. gambiae (Paired Wilcoxon V = 30 p = 0.051, and V = 24, p = 0.024 for An. gambiae and An. arabiensis respectively, Figure 1b). Despite fewer loci being sequenced, this effect could also be detected in An. melas (π_a = 0.15% vs. 0.06%, unpaired Wilcoxon test W = 25, p = 0.034, Figure 1b).

Tajima's D statistic for synonymous sites was negative in both populations of An. arabiensis (D = -0.29 and D = -0.38, averages across loci in TZ and BK respectively) and in An. gambiae BK (D = -0.71, M-form individuals only), and did not differ between serpins and other genes (Wilcoxon tests, p > 0.5 in all cases). Tajima's D did not differ significantly between the two populations of An. arabiensis (Paired Wilcoxon V = 193, p = 0.42), but did correlate between the populations (rho = 0.43 S = 2543.131, p = 0.017). In population BK, Tajima's D was correlated between An. gambiae and An. arabiensis (rho = 0.490 S = 2293, p = 0.006), but was significantly more negative in An. gambiae (Paired Wilcoxon V = 115, p = 0.015). Strikingly, Tajima's D was generally positive in An. gambiae population KY (mean across loci 0.77, 22 out of the 28 genes with non-zero diversity had D > 0, with overall 95% bootstrap interval [0.40, 1.15]). This was significantly higher than in BK (Paired Wilcoxon test V = 15, p = 1 × 10^-6).

In An. arabiensis, five genes had individually significantly negative Tajima's D statistics (p < 0.05 in all cases, no correction for multiple tests): control loci 1 (BK and TZ) and 5 (BK), and serpins 10 (BK), 6 and 7 (TZ). In An. gambiae population BK (M-form only) 7 genes had significantly negative D values: serpins 7, 9 and 14, and control loci 5, 6, 10 and 11 (p < 0.05 in all cases).

In An. arabiensis, differentiation between East and West Africa was very low (K_ST = 0.08) and not significantly different from zero at 15 of the 32 loci examined. In An. gambiae, differentiation between East and West Africa was much higher (K_ST = 0.14; all loci except control locus 9 were individually significantly differentiated) and this difference between the species was statistically significant (paired Wilcoxon test V = 392, p = 0.016). Note that this differentiation reflects not only geographic separation, but also differentiation between M and S molecular forms of An. gambiae. In West Africa (BK) differentiation between M- and S-form was very low (K_ST = 0.016), and significantly lower than differentiation between S-form sampled from East Africa and S-form sampled from West Africa (K_ST = 0.073, paired Wilcoxon test V = 442, p = 0.0005). Only two S-form An. gambiae individuals were sampled from BK, making estimates of differentiation potentially poor and reducing the power of the test. However, assuming random mating, the estimates should not be biased by the small number of individuals sampled, and the large number of loci (32, ~19 Kbp of sequence) will reduce sampling error. For an overview of genetic differentiation see Figure 2.

Anopheles gambiae population KY was highly polymorphic for the 2La/+ chromosomal inversion, allowing us to test for differentiation between the two inversion states. Dividing population KY into two groups on the basis of on inversion status (2La homozygotes versus 2L+^a homozygotes, heterozygotes excluded) identified very strong population structure associated with the inversion. At the six loci sampled from within the inversion (serpins 1–3 and control loci 1–3) mean differentiation between inversion-groups was K_ST = 0.25 (all six loci were significantly differentiated: permutation p < 0.01 for each locus). Across the six other polymorphic loci sequenced from chromosome arm 2L (serpins 7, 10, 14 and 18, control loci 10 and 18), differentiation between these groupings was K_ST = 0.03, and was not significantly different from zero in any locus (p > 0.14 by permutation, in each locus).

Genetic divergence (substitutions per site) between An. gambiae and An. arabiensis was very low: K_S = 3.5% (averaged across loci), and when corrected for diversity (i.e. , page 220 in 54), K_S = 1.2%. No synonymous fixed differences were identified between these species, and only three non-synonymous fixed differences (all in control locus 5, derived from a transposable element). Divergence from An. melas was higher for both An. gambiae (K_S = 6.4%, corrected K_S = 4.6%) and An. arabiensis (K_S = 6.3%, corrected K_S = 4.9%), as was divergence from An. merus (uncorrected K_S = 5.3% and K_S = 4.8%, respectively); divergence between these species and An. quadriannulatus was intermediate (uncorrected): K_S = 4.01% and K_S = 4.30% respectively. For an overview of interspecies divergence and an illustration of gene trees versus species trees, see Figure 3.

Genetic differentiation (the proportion of total diversity attributable to between-species differences) between An. gambiae and An. arabiensis species was K_ST = 0.19 (95% bootstrap interval across loci [0.16, 0.24]). Differentiation between An. gambiae and An. melas was much higher (K_ST = 0.33 [0.27, 0.40]), as was differentiation between An. arabiensis and An. melas (K_ST = 0.50 [0.44, 0.57]). Differentiation between An. gambiae and An. arabiensis was lower for serpins than for control loci (K_ST = 0.14 vs. K_ST = 0.25, paired Wilcox test V = 113, p = 0.02). However, although the trend was in the same direction in East and West Africa, this effect was only significant in West Africa (V = 108, p = 0.04, as compared to V = 99, p = 0.12).

Due to the lack of fixed differences between An. gambiae, An. arabiensis and An. quadriannulatus, McDonald-Kreitman based approaches were not applied to these data (see Discussion). However, it was possible to apply the method of Welch 30 using An. gambiae/An. arabiensis, and their divergence from An. melas and/or An. merus. According to the likelihood ratio test, neither analysis using An. melas provided any evidence supporting adaptive substitutions between the lineages (there was no significant improvement in model fit between fixing α = 0 and allowing α to take its maximum likelihood value; p > 0.19 in all cases: Table 2). This was also true for the test using An. gambiae and An. merus (p > 0.2: Table 2). For the test using An. arabiensis and An. merus there was some evidence that α was significantly greater than zero, (α = 0.34 [0.08, 0.53]; 2ΔlnL = 6.16, 1d.f., p = 0.013, significance lost if a correction is made for multiple tests), but no significant improvement in model fit was obtained by allowing α to differ between serpins and control loci, or between Plasmodium-related serpins and all other loci. This suggests that approximately 8–53% of amino-acid substitutions between An. arabiensis and An. merus were adaptive, but this value did not differ significantly between serpins and the control loci. Analysis of Akaike weights gives a qualitatively identical result: in each species-pair no model is strongly preferred, but in the merus-arabiensis comparison, α = 0 receives relatively little weight. Full results of the McDonald-Kreitman analysis are presented in table 2, and raw data are given in Additional file 4.

The An. gambiae complex falls within the Pyretophorus series of the subgenus Cellia, and comprises a closely-related group of approximately eight species (An. gambiae s.s., An. arabiensis, Anopheles bwambae, An. quadriannulatus A and B, An. merus, An. melas and Anopheles comorensis) 555657, plus at least one case of incipient speciation (M and S molecular forms of An. gambiae s.s. 37585960). Because lineages within the complex differ in their importance as Plasmodium vectors [e.g., 61], in their ecological preferences 6263, and in their resistance to pesticides [e.g. 63], there is considerable value in understanding both species relationships and how populations are structured. This may be of particular consequence if any attempt is ever made to genetically modify wild Anopheles populations to block or reduce Plasmodium transmission 6465. However, in addition to having important implications for vector control, as discussed below, understanding phylogeny and population history are also essential to the robust inference of selection.

Previous analyses suggest that An. gambiae and An. arabiensis are sister taxa, and the data presented here from five of the eight species are in strong agreement, placing An. gambiae and An. arabiensis as the most closely related species pair, with greater divergence to An. merus and An. melas (Figure 3a; note that the tree is unrooted). However, in common with previous studies [e.g. 336667], these data suggest extensive shared polymorphism (Figure 3) and very low differentiation between An. gambiae and An. arabiensis (K_ST = 0.19). The inter-species differentiation between An. gambiae and An. arabiensis is approximately the same as inter-population differentiation between African and European D. melanogaster (K_ST = 0.16 to 0.24, depending on population; pers. comm. P. R. Haddrill, data from 68), and is lower than inter-population differentiation in the predominantly selfing nematode Caenorhabditis elegans (K_ST = 0.38, pers. com. A. D. Cutter, data from 69). Thus, although An. gambiae and An. arabiensis are largely reproductively isolated and significantly differentiated 7071, these data confirm that either they share extensive ancestral polymorphism, or that there is considerable introgression between them [see also 677273]. This is further supported by the very high correlation in neutral diversity across genes, between these two species (Additional file 2).

Given a particular divergence time, effective population size is the primary determinant of the amount of shared ancestral polymorphism between taxa, because drift (and therefore lineage-sorting) is faster in small populations [e.g. 74]. Thus, although it is likely that An. gambiae and An. arabiensis share a more recent common ancestor that either does with An. melas, the lower differentiation between An. gambiae and An. arabiensis could also be explained (at least in part) by differences in effective population size within the complex. Since π_s is an estimator of 4N_eμ, differences in neutral diversity imply that An. gambiae and An. arabiensis have larger effective population sizes than An. merus, consistent both with their wider geographic range, and with potentially higher levels of shared ancestral polymorphism (see above: π_s~2.8%, 2.0%, and 0.9% for An. gambiae, An. arabiensis and An. melas, respectively). Diversity in An. gambiae and An. arabiensis is similar to that seen in African populations of Drosophila simulans and D. melanogaster (π_s = 3.2% and π_s = 1.7%, respectively) [e.g. 75], and assuming the mutation rate (μ) is similar between mosquitoes and Drosophila, this also suggests a long-term effective population size for An. gambiae that is about 70% larger than D. melanogaster 76, i.e. well in excess of 1 million. This is broadly consistent with previous estimates from mitochondrial sequence, but is much larger than estimates based on microsatellites and allozyme variants [reviewed in 77].

Incipient speciation between the M and S molecular forms of An. gambiae is a major focus of ongoing research 37585960, culminating in the recently completed sequencing of the M and S molecular-form genomes 78. Although differentiation between the M and S form of An. gambiae in West Africa is extremely low (K_ST = 0.02; Figure 2), it is consistently non-zero at some loci, even where the lineages are sympatric (see e.g. 6). This unambiguously identifies M and S form An. gambiae as being (at least partly) reproductively isolated 58, and it has been argued that different M and S-form niches may be distinguishable 37. Moreover, differentiation is variable around the genome, being higher at so-called 'islands of speciation', potentially associated with adaptive differences 5859. However, other studies have shown that in some geographic regions microclimate is a better predictor of population divergence than is molecular form 79, and it is clear that the S-form of An. gambiae is not a single homogenous lineage 6680. Indeed, it is well-established that there is extensive genetic differentiation associated with the Great Rift Valley 80, and consistent with this, the data presented here not only identify considerable differentiation between East and West Africa (KY S-form versus BK M-form, K_ST = 0.12), but also show that West African M-form and S-form are less differentiated from each other than either is from East African S-form (Figure 2). This suggests either that the West African M and S lineages share a more recent common ancestor, or alternatively that they have experienced considerable recent gene flow [37, e.g. 80, but see also 81]. For An. arabiensis, differentiation across the width of the continent is only K_ST = 0.08, which is very similar to that between Eastern and Western S-form An. gambiae (K_ST = 0.07; Figure 2), and approximately twice that between D. melanogaster populations sampled across a similar geographic range (Gabon vs. Kenya or Zimbabwe, K_ST = 0.04) [P. R. Haddrill pers. comm., data from 68].

Most populations showed a slight skew in the allele-frequency spectrum toward low-frequency variants (i.e., average Tajima's D was negative), consistent either with population growth, or with weak selection against some synonymous variants. In contrast, however, the data presented here also identify a strong skew toward intermediate frequency alleles in An. gambiae population KY (i.e., a positive Tajima's D, 0.77 averaged across loci, 95% bootstrap interval 0.40–1.15). One potential explanation for this is that population KY is admixed or contains cryptic population structure, for example as would be the case if two divergent lineages of S-form are coexisting there. Alternatively, a positive Tajima's D could also result from a recent decrease in effective population size.

The phylogenetic and phylogeographic complexity of the An. gambiae species group means that inferences drawn from single individuals should be treated with caution, as the low differentiation between species and high diversity within species means that any one individual is not necessarily typical or representative [e.g. 66]. For example, the recently sequenced M and S-form genomes 78 were both obtained from mosquitoes sampled in Mali, but S-form divergence between East and West Africa is considerably greater than M-S divergence within West Africa (see Figure 2 above, and 6680, cf. 81). It is therefore not clear that any conclusions regarding M - S genome divergence will generalize to S-form individuals from the east African coast. The same issue arises with inter-species comparisons. For example, the divergence between two randomly sampled An. gambiae genomes is K_S~3%, and that between one randomly selected An. gambiae genome and one randomly selected An. quadriannulatus genome is only K_S~4% (above, and Figure 3). This means that a single inbred strain of An. quadriannulatus is only marginally more informative about An. quadriannulatus than it is about An. gambiae, and without more extensive sampling it cannot reliably be used to identify genetic differences between the species [cf. 61]. These issues will be of paramount importance in analysing the recently approved complete genomes of An. arabiensis, An. quadriannulatus and An. merus 82.

There is considerable evidence from other taxa that serpins are an evolutionarily dynamic gene family, with high turnover between lineages and occasional lineage-specific expansions (e.g. 23, see 13 for a review). For example, although most Anopheles and Aedes serpins have 1:1 orthologs, there are 29 serpins in D. melanogaster but only 18 in An. gambiae, and very few mosquito serpins have 1:1 orthologs in Drosophila 83. Some serpins also show very high rates of adaptive evolution, such as Drosophila Spn28D [CG7219 in ref. 21], and it has been suggested that, in general, rapid turnover and strong selection in serpins may be associated with serpin immune function, and could be driven by an evolutionary 'arms-race' 13. In An. gambiae, three serpins are known to have immune-related function in response to Plasmodium infection (SRPN2, SRPN6 and SRPN10 in 242628).

Strong selection can affect patterns of genetic diversity, both between populations, and between chromosomal inversions. In An. gambiae population KY, considerable differentiation was identified between 2La and 2L+^a homozygotes around the SRPN1, 2 and 3 cluster (and control loci 1, 2 and 3). For these loci K_ST = 0.25 between inversion states, which is actually higher than the overall differentiation between An. gambiae and An. arabiensis, and twice as high as the differentiation between M-form and S-form An. gambiae. No such differentiation was seen for other loci on chromosome arm 2L, indicating that this is strongly associated with the inversion. Although chromosomal inversions are in general expected to suppress recombination, especially near breakpoints, this is unlikely to lead to extreme or long-term differentiation unless maintained by selection [e.g. 8485]. In particular, despite recombination being suppressed in heterozygous individuals, genetic exchange (including recombination and gene-conversion) within the region of the 2La/+ inversion is not zero 86, and this should allow such differentiation to break down rapidly if it is not selectively maintained. The finding of elevated differentiation around these loci agrees with previous analyses, which found the SRPN1-3 cluster to be close to the region of highest differentiation between 2La and 2L+^a. 31. While this doesn't provide strong evidence that any of these serpins are being directly selected, it is interesting to note that SRPN2 is required for successful infection of An. gambiae by P. berghei 28, and that 2La inversion-status was identified with vector-competence in some early studies 8788. Thus it is possible that genetic structuring in serpins 1–3, introduced and maintained by the 2La inversion, may affect variation in vector competence, even if the underlying cause of 2La/+ differentiation is elsewhere.

In contrast, using a McDonald-Kreitman based approach to detecting adaptive substitutions 2930, no strong or consistent evidence of selection could be detected, nor could differences in the rate of adaptive evolution between serpins and other genes (or between immune serpins and other genes; Table 2). Specifically, in three of the four species pairings that were analysed, the rate of adaptive evolution could not be distinguished from zero (Table 2). This may indicate that neither Anopheles immune-related serpins, nor Anopheles serpins as a family, are subject to selection for rapid change, and consequently that all selection acting on these genes is purifying. However, it may also be an artefactual result arising either from limitations of the McDonald-Kreitman framework, or from issues specific to the gambiae species complex (see next section). In particular, the McDonald-Kreitman approach assumes that non-synonymous substitutions can be divided into three classes: strongly advantageous mutations that fix rapidly, strongly deleterious mutations that are rapidly lost, and effectively neutral mutations that drift in frequency 50. If there is also a large class of weakly deleterious mutations that remain polymorphic for an extended period, but are lost by selection in the long term, then this will reduce estimates of α 50.

In agreement with this, a trend toward higher amino-acid diversity was found in serpins, as compared to other genes, in all three species (An. gambiae, An. arabiensis and An. melas; Figure 1b). This could suggest that purifying selection on serpins is weak or intermittent (as compared to purifying selection on other genes), or that there is selection favouring diversity in serpins, such as balancing selection 89. However, if the latter were the case, then one might also expect to find an increase in diversity at linked synonymous sites, and although slightly higher, synonymous diversity in serpins did not differ significantly from other genes (Figure 1a), suggesting that they neither experience long-term selection for increased polymorphism, nor undergo more frequent selective sweeps [e.g. 8]. Moreover, in no population or species did a serpin display the highest or lowest neutral genetic diversity, and there was no clear pattern in the allele frequency spectrum (as measured by Tajima's D statistic) or inter-population differentiation (measured by K_ST) that supports the notion of strong selection acting on Anopheles serpins. These data therefore fail to identify any serpins as candidates for recent strong selection within the gambiae complex.

A null result in tests for selection may also result from low power or model violations associated with the phylogenetic and population history in the An. gambiae complex. Motivated by an interest in identifying targets of pathogen-mediated selection, several studies have now attempted to identify adaptive evolution in immune-related genes from the Anopheles gambiae species complex 56323334909192. However, in stark contrast to almost identical studies in taxa such as Drosophila [for example 101121939495], to date there is very little clear evidence supporting adaptive evolution in the An. gambiae complex [but see 9092].

One likely reason for the difference between Anopheles and Drosophila studies is the dearth of suitable outgroups for An. gambiae [e.g. 32]. First, well-studied Anopheles species outside of the gambiae complex appear to be too distantly related to reliably infer the divergence between them. For example, the An. stephensi SRPN6 cDNA sequence 26 suggests that K_S between with An. stephensi and An. gambiae is ~1.33 (± 0.157) substitutions per site when estimated by maximum likelihood 96, or 0.92 (± 0.117) by the method of Li 97. Second, low divergence within the complex means there is little power to infer the substitution rate between them 63233. For example, across all 8 serpins appearing in the extensive survey of Cohuet et al. 6, there are only two fixed amino acid differences between An. arabiensis and An. gambiae. Similarly, the present study found no fixed differences at all in the same genes, probably because wider geographic sampling provided greater power to distinguish between polymorphisms and fixed differences. The stochastic errors involved here mean that estimates of substitution rate are likely to be wildly variable, reducing the power to estimate the fraction of adaptive substitutions using a McDonald-Kreitman framework (Figure 4).

In principle, other species from within the gambiae complex might be informative outgroups for An. gambiae and An. arabiensis: although divergence is small (1 to 5%; Figure 3a), this may be sufficient in other taxa, such as the human-chimp comparison [e.g. 98]. However, unlike the human-chimp case, diversity in Anopheles is very high compared to humans (π_s~3% in An. gambiae, ~2% in An. arabiensis, Figure 1, c.f. ~0.1% in humans, e.g. 99), and this leads to two potential problems. First, a large proportion of apparent substitutions will in reality be polymorphisms 3051, and although this effect is small enough to be negligible for pairs of species in which K_S >> π_S, it becomes a concern when comparing An. gambiae to An. arabiensis, for which K_S <π_S (Figure 3). However, as here, this effect can be accounted for using models which include the sample size and diversity, and thereby infer the 'true' number of substitutions 3051. Second, and potentially more serious, is the opportunity for extensive shared polymorphism. The McDonald-Kreitman framework uses information from current diversity (i.e. π_s and π_a) to infer whether some proportion of historic substitutions (K_A) cannot be explained by purely neutral processes. This model implicitly assumes a time period when the two lineages were diverging from their common ancestor, during which selection (that is to be detected) was able to act. If there is extensive shared polymorphism between the species, for example if gene trees are rarely reciprocally monophyletic (as is the case with An. gambiae and An. arabiensis; see Figure 3b–d and e.g. 33), then it is hard to see how the McDonald-Kreitman approach can ever be usefully applied. In other words, unlike the straight-forward cases where divergence is too high (the branch is too long) or divergence is too low (the branch is too short), for An. gambiae and An. arabiensis, in effect there is no branch at all (Figure 3b–d).

Unfortunately, for similar reasons, there are also serious concerns about the application of other selection-inference methods to the gambiae complex, such as the phylogenetic methods implemented in PAML 96 and HyPhy 100. These methods use multiple sequences related by a gene (or species) tree to infer relative rates of synonymous and non-synonymous substitution, allowing variable rates at different sites or in different parts of the gene. Most phylogenetic methods assume there is no recombination within loci [but see OmegaMap, 101], and simulation suggests false positives can reach >50% when 2N_er > 0.01 102. Because 2N_er (i.e: 2 × effective population size × recombination rate per codon per generation) in An. gambiae is likely to be of the order 0.01 – 0.1 or higher – primarily due to the large effective population size – such phylogenetic approaches cannot be applied reliably to within-species Anopheles data.

Additionally, where the McDonald-Kreitman framework assumes that between-species K_A results from the joint action of selection and drift, and within-species π_a results only from drift, the phylogenetic approaches (as they are most commonly applied) assume either that all amino acid variants have the same cause (i.e. K_A and π_a do not provide independent information about selection and drift) or that most differences are fixed between species, (i.e. K_A >> π_a, such that π_a is negligible). Thus the phylogenetic and McDonald-Kreitman approaches constitute very different models that lend themselves to different datasets, and it is not clear that any single dataset can reasonably be analysed using both. Instead, to analyse a dataset that includes substantial within-species sampling using a phylogenetic approach, it may be more rational to fit a model which allows the relative rates of synonymous and non-synonymous substitution to differ between within- and between-species branches [e.g. 103]. However, in the case of the gambiae complex, the low power associated with the low inter-species divergence will then be encountered again.

If McDonald-Kreitman tests have relatively low power within the gambiae complex, and phylogenetic methods cannot easily be applied to within-species data, how can selection be inferred from Anopheles population genetic data? One possibility is to use approaches based solely on within-population diversity to identify recent selective sweeps or regions of elevated polymorphism, and this will work for some loci [e.g. TEP1, 92], though the possibility of introgression, and/or chromosomal inversions that might affect the distribution of diversity, should then be taken into account. Nevertheless, at present it seems the best options for outgroup-based analyses are An. merus and/or An. melas, and both the data presented here (Figure 3) and previous studies [e.g. 3366] suggest that their divergence from the An. gambiae/An. arabiensis clade should be sufficient in the case of genes evolving under very strong selection. However, the recently approved genome sequences from 13 more species of Anopheles mosquitoes 82 may hold the solution, and particularly the complete genome sequence of Anopheles sundaicus (also subgenus Cellia, Pyretophorous series).