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Breakdown in the Process of Incipient Speciation in Anopheles gambiae

Breakdown in the Process of Incipient Speciation in Anopheles gambiae INVESTIGATION Breakdown in the Process of Incipient Speciation in Anopheles gambiae † ‡ Davis C. Nwakanma,* Daniel E. Neafsey, Musa Jawara,* Majidah Adiamoh,* Emily Lund, § †† ‡‡ §§ Amabelia Rodrigues, Kovana M. Loua,** Lassana Konate, Ngayo Sy, Ibrahima Dia, †,‡,††† ,‡‡‡,1 T. Samson Awolola,*** Marc A. T. Muskavitch, and David J. Conway* † ‡ *Medical Research Council Laboratories, Banjul, The Gambia, Broad Institute, Cambridge, Massachusetts 02467, Harvard School of ** Public Health, Boston, Massachusetts 02115, Bandim Health Project, Bissau, Guinea-Bissau, National Institute of Public Health, †† ‡‡ Conakry, Republic of Guinea, Universite Cheikh Anta Diop, Dakar, Senegal, Service de Lutte Antiparasitaire, Thies, Senegal, §§ ††† *** Institut Pasteur, Dakar, Senegal, Nigerian Institute of Medical Research, Yaba, Lagos, Nigeria, Boston College, Chestnut ‡‡‡ Hill, Massachusetts 02467, and London School of Hygiene and Tropical Medicine, London, WC1E 7HT United Kingdom ABSTRACT Understanding genetic causes and effects of speciation in sympatric populations of sexually reproducing eukaryotes is challenging, controversial, and of practical importance for controlling rapidly evolving pests and pathogens. The major African malaria vector mosquito Anopheles gambiae sensu stricto (s.s.) is considered to contain two incipient species with strong reproductive isolation, hybrids between the M and S molecular forms being very rare. Following recent observations of higher proportions of hybrid forms at a few sites in West Africa, we conducted new surveys of 12 sites in four contiguous countries (The Gambia, Senegal, Guinea- Bissau, and Republic of Guinea). Identification and genotyping of 3499 A. gambiae s.s. revealed high frequencies of M/S hybrid forms at each site, ranging from 5 to 42%, and a large spectrum of inbreeding coefficient values from 0.11 to 0.76, spanning most of the range expected between the alternative extremes of panmixia and assortative mating. Year-round sampling over 2 years at one of the sites in The Gambia showed that M/S hybrid forms had similar relative frequencies throughout periods of marked seasonal variation in mosquito breeding and abundance. Genome-wide scans with an Affymetrix high-density single-nucleotide polymorphism (SNP) micro- array enabled replicate comparisons of pools of different molecular forms, in three separate populations. These showed strong differentiation between M and S forms only in the pericentromeric region of the X chromosome that contains the molecular form- specific marker locus, with only a few other loci showing minor differences. In the X chromosome, the M/S hybrid forms were more differentiated from M than from S forms, supporting a hypothesis of asymmetric introgression and backcrossing. HE major malaria vector mosquito Anopheles gambiae restricted to western parts of Africa, and hybridization between Tsensu stricto (s.s.) exists throughout most of Sub-Saharan them is rare in most areas of sympatry (Della Torre et al. Africa, but there are many polymorphisms including chro- 2005). Scanning of large numbers of polymorphic markers mosomal inversions that appear to be involved in the adap- initiallyindicated themolecular formswereveryhighlydiffer- tation of subpopulations to different environments (Coluzzi entiated at only a small number of discrete regions within the et al. 2002), and molecular forms (M and S) have been iden- two autosomes and the pericentromeric region of the X chro- tified that appear to be reproductively isolated (della Torre mosome, which contains the form-specific markers in the ribo- et al. 2001). The S form is distributed widely throughout the somal (r)RNA genes (Turner et al. 2005). However, a finding A. gambiae species range, whereas the M form is common but that transposable element insertion sites showed marked dif- ferentiation between the forms indicated that reproductive iso- lation might affect a larger portion of the genome (Esnault Copyright © 2013 by the Genetics Society of America doi: 10.1534/genetics.112.148718 et al. 2008). A high-density array to type 400,000 single- Manuscript received September 19, 2012; accepted for publication January 13, 2013 nucleotide polymorphisms (SNPs) has recently enabled more Available freely online through the author-supported open access option. thorough genome-wide analyses, confirming that the molec- Supporting information is available online at http://www.genetics.org/lookup/suppl/ doi:10.1534/genetics.112.148718/-/DC1. ular forms are differentiated at many loci throughout the Corresponding author: Department of Pathogen Molecular Biology, London School of genome in addition to those previously shown (Lawniczak Hygiene and Tropical Medicine, Keppel St., London WC1E 7HT, United Kingdom. E-mail: [email protected] et al. 2010; Neafsey et al. 2010; Reidenbach et al. 2012). Genetics, Vol. 193, 1221–1231 April 2013 1221 It is vital to understand such differentiation and genetic subdivision and its importance in vector evolution, as this complexity can affect malaria control, including resistance to insecticides (Dabire et al. 2009; Djogbenou et al. 2010; Lynd et al. 2010) and susceptibility to malaria parasites and other infections (Rottschaefer et al. 2011; White et al. 2011). Differences in the adult mosquito transcriptome be- tween the M and S molecular forms appear to be minimal (Cassone et al. 2008; Aguilar et al. 2010), but evidence is accumulating that the larval stages are differentially adap- ted to particular features of breeding sites, with the M forms being generally more common in large areas of irrigation for crop cultivation (Diabate et al. 2008; Gimonneau et al. 2011). Studies of mosquito behavior suggest assortative mating may be the means of preventing natural hybridiza- tion between the forms, as most mating swarms consisted of single forms in an area of sympatry in Mali (Diabate et al. 2009), although some mixed swarms were seen in Burkina Faso (Diabate et al. 2006), and very close-range barriers Figure 1 Map of sampling locations for surveys of Anopheles gambiae s.s. molecular forms between 2007 and 2009 in The Gambia, Senegal, to interbreeding may also operate (Pennetier et al. 2010; Guinea-Bissau, and Republic of Guinea. Each location is indicated by Sanford et al. 2011). a yellow circle, and the frequencies of the M form, the S form and M/S Surveys of populations in the extreme west of Africa show hybrid forms sampled at each are indicated by an adjacent pie chart substantial local variation in relative frequencies of A. (sample sizes and exact frequencies are given in Table 1). gambiae s.s. M and S molecular forms (della Torre et al. 2005). For example, the M form predominates in most of employed a pooled hybridization approach for expediency, The Gambia, but ,100 km farther inland in eastern Senegal allowing use of a genotyping array with much higher marker almost all members of the species are S form (Caputo et al. density than that of previous surveys for introgression in West 2008). Of particular interest are areas in which both forms Africa (Marsden et al. 2011; Weetman et al. 2012). Results exist, as the occurrence of natural hybrids may be possible. show extensive hybridization throughout the area and that Elsewhere in Africa, areas of sympatry consistently exhibit mosquitoes with M and S form-specificmarkers are largely very low frequencies of hybrid forms, but a few years ago homogenized throughout the genome, with evidence support- exceptionally high proportions of M/S hybrids were reported ing a hypothesis of asymmetric hybridization and frequent at a few sites near the west coast, with 3% at Dielmo in backcrossing particularly between the M/S hybrid forms and Senegal (Ndiath et al. 2008), 7% at Njabakunda in The Gam- the S forms. bia (Caputo et al. 2008), and 24% at Antula in Guinea-Bissau (Oliveira et al. 2008). Analysis of an insertion polymorphism linked to the form-specific marker on the X chromosome, Materials and Methods as well as an unlinked SNP on chromosome 3, in a subset of Sampling sites for A. gambiae mosquitoes the samples from The Gambia and Guinea-Bissau suggested that there may be introgression between the forms (Caputo Twelve sites were surveyed in four countries (Figure 1). These et al. 2011), and separate studies using broader scans of ge- were chosen to encompass the few areas where unusually high nomic polymorphisms in samples from Guinea-Bissau have frequencies of M/S hybrids had previously been noted and to supported this inference (Marsden et al. 2011; Weetman extend the sampling to the north and south of these, within et al. 2012). 100 km of the west coast of Africa. The northern part of the To thoroughly discover and map the area with high sampling range has Sudan Savannah-type vegetation, with frequencies of hybrids and assess its impact on genomic most annual rainfall of 600–1000 mm normally occurring differentiation, we conducted new surveys of diverse sites in within 4 months (June to September). The south of the sam- the coastal areas of four contiguous countries (The Gambia, pling range has Guinea Savannah-type vegetation including Senegal, Guinea-Bissau, and Republic of Guinea). To in- more woodland, with annual rainfall of up to 2000 mm extend- vestigate the stability of molecular form and hybrid frequen- ing over a longer season (May to October). In all areas, the cies over time throughout different seasons, we conducted majority of A. gambiae s.s. breeding occurs during and at the a longitudinal survey over a 2-year period in the Njabakunda end of the annual rains, and highest densities of adult mosqui- area of The Gambia. We performed high-density genome- toes are found between August and October. The 12 sites are wide scans for differentiation between sympatric M and S numbered and described below (sites 1 and 2 represent Sudan forms, as well as M/S hybrids, at the Njabakunda site and Savannah, sites 3–6 represent northern Guinea Savannah, and at single sites from each of Senegal and Guinea-Bissau. We sites 7–12 represent southern Guinea Savannah). 1222 D. C. Nwakanma et al. Table 1 Proportions of M/S hybrid heterozygote forms and M and S homozygote forms of Anopheles gambiae s.s.in each of 12 sites surveyed from four West African countries Country Site No. mosquitoes M S M/S H F exp IS Gambia Njabakunda 1474 0.164 0.760 0.076 0.322 0.76 Senegal Madina Djikoye 77 0.325 0.350 0.325 0.500 0.35 Senegal Bounkiling 145 0.683 0.138 0.179 0.354 0.49 Senegal Bignona 30 0.767 0.133 0.100 0.295 0.66 Senegal Marsassoum 434 0.270 0.387 0.343 0.493 0.30 Senegal Sedhiou 389 0.653 0.211 0.136 0.402 0.66 Guinea-Bissau Caio 12 0.417 0.167 0.417 0.469 0.11 Guinea-Bissau Antula 464 0.194 0.470 0.336 0.461 0.27 Guinea-Bissau Prabis 91 0.363 0.385 0.253 0.500 0.49 Guinea-Bissau Buba 194 0.907 0.041 0.052 0.130 0.60 Guinea-Bissau Mansoa 152 0.467 0.322 0.210 0.490 0.57 Guinea Boke 37 0.622 0.270 0.108 0.435 0.75 H , expected proportion of M/S heterozygotes under Hardy–Weinberg equilibrium, assuming random mating. There was a significant hetero- exp zygote deficiency (P , 0.005) in each of the populations except for Caio. F inbreeding coefficient (proportion of heterozygotes missing compared IS, to expectations under random mating). The Gambia: Site 1, the Njabakunda village area (13339N, (11489N, 15449W) is a village in a peri-urban area of Bissau 15549W) in the North Bank region of the country (30 km with marsh vegetation and rice cultivation, (site 10) Mansoa west of Farafenni town), was sampled, as previous surveys (1249N, 15199W) is a small town in the north of the coun- (in 2005 and 2006) had shown this area to contain a higher try in a rice cultivation area, and (site 11) Buba (11359N, frequency of M/S form hybrids than elsewhere in The Gam- 1509W) is in the south of the country on the River Rio bia (Caputo et al. 2008). The site is 4 km away from the Grande de Buba near the National Park Contanhez contain- Gambia River on free-draining laterite soil, covered with ing a canopy of tropical rain forest, within which the village open woodland savannah and farmland mainly for cultiva- of Banta Furu was sampled. These sites were sampled in tion of subsistence and cash crops. Four hamlets (Maria August 2009, and Prabis was also sampled in October 2008. Samba Nyado, Sare Illo Buya, Kerr Birom Kardo, and Kerr Sama Kuma) around Njabakunda and within 1 km of each Republic of Guinea: One area in Guinea was sampled in other were selected for longitudinal sampling for a 2-year August 2009, to the south of the border with Guinea-Bissau: period between April 2007 and March 2009. Fortnightly (site 12) near Boke town (10569N, 14189W) four villages (mid-month and end of the month) collections were con- (Korera, Dabaya, Balangdougou, and Bintoumodia) in ter- ducted from June to December covering the rainy season rain of flat plains interspersed with hilly slopes divided by period, and monthly (end of the month) collections were fast-flowing streams, fed by a higher rainfall than at any of done from January to May covering the dry season. the other sites studied here. Collection and identification of mosquitoes Senegal: Five sites were sampled in Senegal. One of these Mosquitoes were sampled from houses or inhabited huts in (site 2), Madina Djikoye (clustered with nearby villages each site, using indoor pyrethrum spray collections (PSC) in Kerr Samba Gueye and Kerr Ousainou Dieng) (13379N, 16189W) is in the Kaolack region immediately to the north most cases for all the sites, with some sampling by indoor of the Gambian border. The other four sites are south of overnight light trap collections (LTC) in addition to PSC in The Gambia in the lower Cassamance region: (site 3) Sed- Guinea-Bissau and Republic of Guinea sites. All mosquitoes hiou (12439N, 1549W) adjacent to the Cassamance River, that were collected were identified morphologically in the (site 4) Bounkiling (1329N, 15429W), (site 5) Bignona field, and A. gambiae sensu lato specimens were stored indi- (12479N, 16149W),and (site6)Marsassoum(12509N, vidually in 1.5-ml polyethylene tubes with desiccant or in 15589W). The sites have free-draining laterite sand or al- Carnoy’s solution if they were semigravid, for subsequent luvial soil and are covered with open woodland savannah DNA typing of species and molecular forms. DNA was ex- or farmland for groundnut and cereal cultivation. These tracted from individual mosquito specimens, using the Corbett sites were sampled in August 2009, except for Sedhiou, Robotics X-Tractor Gene automated DNA extraction platform which was sampled in October 2008. (QIAGEN, Crawley, UK) according to the manufacturer’s protocol for tissue extraction. Whole mosquito material was Guinea-Bissau: Five sites were sampled in Guinea-Bissau: extracted, except for a few legs of each specimen retained for (site 7) Caio village (11559N, 16129W) is in a coastal area confirmatory PCR testing in case the initial result indicated of rice and cashew plantations mixed with woodland a hybrid molecular form. Each of the three species of the A. with traditional ritual significance, (site 8) Antula (11539N, gambiae complex that exist in West Africa (A. gambiae s.s., A. 15359W) is a village east of the capital Bissau, (site 9) Prabis arabiensis,and A. melas) were discriminated in the laboratory Interbreeding Between Incipient Species 1223 Figure 2 Numbers of molecular forms M and S and M/S hybrid forms sampled in each month from a total of 1474 Anopheles gambiae s.s. individuals collected in the Njabakunda area of The Gambia over a 2-year period from April 2007 until March 2009. by species-specificPCR of sequenceswithinthe rRNA gene individual hybridizations and pooled hybridizations for pools locus on the X chromosome (Scott et al. 1993), and A. gambiae of 20 individuals (Pearson’s r =0.96, Supporting Informa- s.s. mosquitoes were differentiated into molecular forms M tion, Figure S1), leading to the repetition of that assay design and S (and M/S hybrids) by form-specific restriction enzyme here. Following Robust Multi-array Average (RMA) back- digestion of the amplified fragment (Fanello et al. 2002). ground correction and quantile normalization using Affyme- trix Power Tools, allelic balance at assayed loci within the Genome-wide scans for differentiation between pools pools was inferred using the Contrast statistic generated by of A. gambiae s.s. mosquitoes BRLMM-P, using a K value of 1 (Rabbee and Speed 2007). A custom Affymetrix oligonucleotide array for genome-wide Such plots result in a modal background mean Contrast dif- typing of 400,071 A. gambiae s.s. SNPs (Neafsey et al. 2010), ference of 0.25 rather than 0, which is the result of noise in termed the 400K array, was used for population genotyping. the signal rather than a genome-wide divergence signal. Plots DNA hybridization was performed with separate pooled of local divergence along the chromosomes were generated samples of 20 homozygous M-form and 20 homozygous using a stepping window approach, by calculating the mean S-form A. gambiae s.s. mosquitoes from each of three sites of the absolute value of the arithmetic difference in Contrast sampled (Njabakunda in The Gambia, Sedhiou in Senegal, for 50 adjacent assays. The mean physical distance spanned and Antula in Guinea-Bissau). The Njabakunda site was the by 50 adjacent assays was 28.2 kb (range = 9.1–280 kb, primary one selected for this genome-wide SNP analysis, as standard deviation = 16.6 kb). Thresholds for statistical sig- we studied this site intensively. Therefore, it had the largest nificance (a = 0 0.05 after Bonferroni correction) for these sample size and most accurate estimation of frequencies. We windowed analyses were calculated using a bootstrapping added the other two sites to include one from Senegal and approach, in which mean Contrast difference was calculated one from Guinea-Bissau, each of which had large sample for each pairwise comparison for 50 SNPs that were sampled sizes with higher proportions of hybrids. In addition, a pool randomly from the genome a total of 10 million times. of DNA from 20 M/S hybrid mosquitoes was tested and compared with the homozygous samples, for each of these Results three sites. Each of the 180 individual mosquitoes that con- Quantifying proportions of molecular forms and hybrids tributed to the pooled arrays (20 M form, 20 S form, and at different sites 20 M/S forms, selected from each of the three sites noted) was also genotyped for the SINE200x6.1 transposon insertion Molecular form typing was performed on a total of 3499 A. site polymorphism that is linked to the molecular form marker gambiae s.s. mosquitoes sampled from the 12 sites surveyed in on the X chromosome (Santolamazza et al. 2011). Affymetrix the four countries (Senegal, The Gambia, Guinea-Bissau, and oligonucleotide array hybridization was performed following Republic of Guinea) (Figure 1). In each site, M and S molecular methods already described (Neafsey et al. 2010), as previous forms coexisted sympatrically and substantial proportions of analysis revealed a very high correlation between aggregate M/S hybrid forms were found (from 5 to 42%) (Table 1). The 1224 D. C. Nwakanma et al. Figure 3 Test for genomic differentiation between pooled DNA from samples of 20 homozygous M-form and 20 homozygous S-form Anopheles gambiae s.s. from each of three sites in this study: (A) Njabakunda in The Gambia, (B) Sedhiou in Senegal, and (C) Antula in Guinea-Bissau. The vertical axis represents divergence measured as the absolute value of mean Contrast difference for stepping windows of 50 assays (this has a modal background of 0.25 that is due to noise rather than a genome-wide divergence signal), using data from all SNPs on the 400K genome-wide array. The horizontal blue lines indicate the Bonferroni- corrected threshold for statistical significance obtained via bootstrapping. Only the X-chromosomal pericentromeric region that contains the M and S molecular form markers exhibits differentiation. For comparison, D shows differentiation between similarly pooled DNA from M and S forms in Mali (Neafsey et al. 2010). estimated inbreeding coefficients for this rRNA gene locus, portions of M/S hybrids were less than expected under Hardy– which has previously been considered a putative speciation Weinberg equilibrium with complete panmixia, but in some marker, showed a wide spectrum of values (from 0.11 to 0.76) cases not much less. (Table 1). Seven of the sites each had samples of .100 mos- Analysis of proportions of molecular forms and hybrids quitoes analyzed, allowing highly accurate estimations of at one site throughout a 2-year period frequencies, and we note that these also show a wide range of M/S hybrid proportions (from 5 to 34%) and estimated To investigate the stability of frequencies over time, we sampled inbreeding coefficients (from 0.27 to 0.76). Thus, the pro- the Njabakunda site in The Gambia throughout a 2-year period Interbreeding Between Incipient Species 1225 Table 2 Number of 50-SNP windows exhibiting significant (P , 0.05) mean contrast differences according to pairwise pool comparison and chromosomal location 2R: 2L: 3R: 3L: X: Chi-square P-value: Total windows n = 2256 n = 1846 n = 1692 n = 1374 n = 829 X vs. autosomes (compared with Mali) Gambia M vs. S 3 2 2 1 42 1.70 · 10 Senegal M vs. S 9 3 1 0 98 7.17 · 10 Guinea-Bissau M vs. S 0 0 0 0 58 7.20 · 10 Mali M vs. S 20 30 21 11 112 Comparator Gambia M vs. M/S 5 3 1 0 37 1.83 · 10 Gambia S vs. M/S 0 1 0 0 0 Not applicable (annual periods extending from April to the following March to other African populations (Figure 3D shows published data capture full seasonal dynamics due to rainfall), yielding 1474 from Mali for comparison). A summary of the number of typed mosquitoes from this site (1048 in the first annual period nonoverlapping windows of 50 adjacent SNPs from each and 426 in the second). The relative frequencies of M/S hybrid chromosomal arm exhibiting significant differentiation in forms were stable in both years, accounting for 8.3% in the first each pairwise comparison is presented in Table 2. M vs. S and 5.6% in the second year (chi-square test, P = 0.07) (Figure comparisons for each of the three separate sites exhibit a sig- 2). During each year, S-form mosquito numbers peaked early in nificantly higher ratio of divergent windows on the X chro- the rainy season, and the peak of the less abundant M form mosome compared to the autosomes, relative to the more occurred later in the season, but M/S hybrid forms were canonical interform divergence profile exhibited by the seen throughout. In 2007, M/S forms had seasonally Mali comparison (chi-square tests on the ratio at each stable frequencies of 8.6% (32/374) in April–July and of the sites compared with Mali: P = 1.7 · 10 for the 8.1% (50/620) in August–October (chi-square test, P = Gambian site and P , 10 for the sites in Senegal and 0.78), while proportions of M forms increased from 1.9% Guinea-Bissau) (Table 2). (Table S1 includes the Contrast (7/374) to 22.6% (140/620) during those respective peri- Difference datafile). ods (chi-square test, P , 10 ); in 2008, M/S forms had To test whether minor signals of differentiation may have frequencies of 4.9% (9/184) in April–July and 5.9% (12/ been being obscured by our windowed analysis approach, 205) in August–October (chi-square test, P =0.67),while we examined the region between 28 and 42 Mb on chro- proportions of M forms increased from 8.1% (15/184) to mosome 2R in closer detail, at the level of individual SNP 36.6% (75/205) in those periods (chi-square test, P = assays, for the comparison between M- and S-form pools in 10 ). Guinea-Bissau (Figure S2). This region was focused on as it contains loci that showed differentiation between the M and Genome-wide scans for differentiation between S forms in a recent study, using a panel of several hundred molecular forms within sympatric samples SNPs with a sample from Guinea-Bissau (Weetman et al. To identify whether marked genomic differentiation be- 2012). Although some individual SNP assays in the region tween M and S forms could be seen, we randomly selected showed high Contrast differences between the pools, the 20 homozygous M-form and 20 homozygous S-form Contrast distributions were similar to those observed in mosquitoes from the Njabakunda population for a pooled- a control region of chromosome 3R (19–32 Mb) and much population genome-wide scan of single-nucleotide polymor- lower than those observed in the highly differentiated X phism differentiation, using the custom Affymetrix 400K centromeric region (Figure S2). To test whether signals of array. Remarkably, with the clear exception of the X chro- differentiation might have been obscured by noise due to mosome pericentromeric region that contains the specific poorly performing SNP genotyping on the array, we re- polymorphism used to stratify the molecular forms, there peated the genome-wide analysis with a subset of 65,974 were no loci that exhibited strong differentiation between SNPs that had previously been validated and shown to be in the M and S molecular forms (Figure 3A). To replicate the Hardy–Weinberg equilibrium for individual molecular forms test, genome-wide comparison between M and S forms in Mali (Neafsey et al. 2010). This confirmed that, outside of was conducted for pools of 20 M-form and 20 S-form the pericentromeric X-chromosomal region, there was no ma- mosquitoes from each of two other sites that also exhibit jor differentiation between the forms in the populations stud- high frequencies of M/S hybrids, at Sedhiou in Senegal ied here, in contrast to the interform differentiation previously seen in Mali (Figure S3). We cannot fully reconcile this obser- (Figure 3B) and Antula in Guinea-Bissau (Figure 3C). Both vation with reports of very narrow regions of differentiation of these sites similarly showed a lack of substantial differen- tiation except in the X-chromosomal pericentromeric region. on 2R in another population sample from Guinea-Bissau This contrasts strongly with the observation of differentia- (Weetman et al. 2012), as differences in findings between tion at pericentromeric autosomal loci and elsewhere the studies may result from fine differences in spatial or tem- throughout the genome of M and S forms sampled from poral sampling or in the sensitivity of the respective assays. 1226 D. C. Nwakanma et al. resultsthatwereconcordantwiththe molecularformtyp- ing in 159 (88%) of the individuals. While there was 98% (59/60) concordance for the M form and 100% (60/60) for the S form, confirming appropriate sample selection and robust comparison of the forms here, the lower con- cordance of 67% (40/60) observed for the M/S forms con- firms that many of these are not F hybrids, but that backcrossing must have occurred over multiple genera- tions. The profile of windows with significant differentia- tion in M vs. M/S comparisons is highly similar among the three countries sampled (Figure 5). Across the pairs of countries, M vs. M/S comparisons exhibit Spearman’s rank correlations ranging between 0.77 and 0.84, whereas S vs. M/S comparisons have Spearman’s rank correlations of only 0.15–0.42 between the same pairs (Table S1). Discussion It is widely considered that the major malaria vector A. gambiae s.s. is undergoing a process of speciation into at least two reproductive units defined as the M and S molec- Figure 4 Genome-wide comparison of an M/S pool of 20 hybrid form mosquitoes with M and S homozygous pools from Njabakunda in The ular forms (della Torre et al. 2001). Although the divergence Gambia, using data from all SNPs on the 400K genome-wide array: (top) of these forms is likely proceeding in many parts of west and M homozygotes vs. M/S hybrid forms; (bottom) S homozygotes vs. M/S central Africa (Reidenbach et al. 2012), our results and hybrid forms. The more complete lack of differentiation between S and those of other recent surveys of populations in the extreme M/S suggests asymmetric gene flow between the forms or selection to west of Africa (Caputo et al. 2011; Marsden et al. 2011; maintain the residual differentiation in the M form. (Replicate compari- sons between the pooled hybrid forms and each of the major forms are Oliveira et al. 2008; Weetman et al. 2012) indicate that shown for two separate populations in Figure S4.) hybridization is common. A contiguous region is now out- lined, covering ecologically diverse sites sampled in a contig- uous area within 100 km of the West African coast within Asymmetric minimal differentiation between hybrid four countries in a zone of ,400 km from north to south, forms and homozygous molecular forms where frequencies of M/S hybrid forms are consistently We also performed pooled array hybridizations, using 20 much higher than elsewhere, and there is minimal interform mosquitoes genotyped as M/S heterozygote “hybrid forms” genomic differentiation outside of the pericentromeric re- from each site and compared these with the M and S homo- gion of the X chromosome. This contrasts significantly with zygote pools. Results for the Gambian population sample the results obtained from populations farther east, using the (Figure 4, Table 2) show no divergence in the X pericentro- same methods (Reidenbach et al. 2012), and is important meric region between S and M/S, whereas some divergence for understanding evolution and implementing control of remains between M and M/S. The degree of differentiation this malaria vector and for investigation of the processes observed in the X pericentromeric region (megabases 15– of speciation. 24.2) is significantly higher in the M vs. M/S comparison A wide range of proportions of M/S hybrid forms was relative to the S vs. M/S comparison (Wilcoxon’s sign-rank detected within both Guinea-Bissau and Senegal, with the test, V = 25,996, P = 2.2 · 10 ). Replication of these Gambian site in the north and the Guinean site in the south comparisons for samples from Sedhiou in Senegal and both showing lower proportions than many of the interven- Antula in Guinea-Bissau showed similar results (Figure ing sites. It appears likely that the area we have surveyed, S4), except that the S and M/S pools showed some detect- extending across these four countries, probably covers most able X pericentromeric differentiation in Senegal (less of the current geographical range of the A. gambiae s.s pop- than observed for the comparison of M and M/S). This ulations that exhibit extensive hybridization. Surveys imme- suggests that although the forms are now highly homoge- diately to the south in Guinea have shown mostly S-form nized, the speed of the process has been asymmetric, with mosquitoes near the coast, and both M and S forms, with partial isolation or a lower recombination rate allowing a very low frequency of hybrids, further inland (della Torre maintenance of greater X chromosome pericentromeric di- et al. 2005; Vezenegho et al. 2009; Carnevale et al. 2010). In vergence in the M form. Genotyping of the SINE200 trans- northern Senegal, A. gambiae s.s. is less common than the poson insertion site polymorphism in the 180 mosquitoes related vector species A. arabiensis and exists mostly as the contributing to the pooled arrays (20M form,20S form, M form (della Torre et al. 2005; Dia et al. 2008), while and 20 M/S forms, in each of the three selected sites) gave surveys in the eastern part of Senegal have shown the S Interbreeding Between Incipient Species 1227 form to be more abundant than the M form, with hybrid forms very rarely detected (della Torre et al. 2005; Caputo et al. 2008), as has been observed in Mali and further east- ward (della Torre et al. 2005; Diabate et al. 2009; Aboagye- Antwi et al. 2010). It is possible that our results reflect consequences of the reversal of a previous process that promotes speciation, with the genomic integrity of one or both of the molecular forms now being substantially compromised by introgression within these western populations. It was previously sugges- ted that areas might exist within which there is extensive gene flow between the molecular forms of A. gambiae s.s., while reproductive barriers are maintained at other sites (Black and Lanzaro 2001; Lehmann et al. 2003). A more recent view, also supported by pooled array hybridizations, is that sporadic bouts of hybridization mediated by changing ecological conditions or a very low background rate of gene flow between M and S forms could be responsible for the relative homogeneity of M and S genomes outside of the “speciation islands” (Reidenbach et al. 2012). Such pro- cesses would not be expected to disrupt the divergence at well-established pericentromeric islands of speciation, however. Our results enable sites to be specifically selected for population genomic studies to investigate mechanisms of reproductive isolation. Studies of male mating swarm composition and behavior will be important to conduct in these areas, to identify whether there are proximal determi- nants of hybridization (Diabate et al. 2006, 2009). Further sampling and measurement of multiple ecological parame- ters at different times of the year are also needed to char- acterize these and additional sites more fully, which could enable exploration of correlations between environmental variables, population genetic structure, and reproductive isolation. If the M and S forms were undergoing genome-wide reciprocal introgression, it is expected that the pericentro- meric divergence on the X chromosome should be gradually eliminated in both forms by recombination. The observation that the X pericentromeric region remains relatively distinct in M-form populations in this area, as also reported recently by others sampling in Guinea-Bissau (Marsden et al. 2011), supports a hypothesis that partial reproductive isolation or selective pressure preserves a narrow genomic island linked to the M-form-specific marker (Caputo et al. 2011; Marsden et al. 2011; Weetman et al. 2012). A high degree of similar- ity in the specific pericentromeric divergence profile among sites (Figure 5, Table S2) further indicates the likely role of Figure 5 Comparison of M vs. M/S divergence among geographic sites in the X pericentromeric region (megabases 15–24.2). Each point represents a 50-SNP window. The profile of differentiation is maintained in a highly similar manner in all pairwise comparisons of geographic sites: (top) Gambia vs. Guinea-Bissau, (middle) Senegal vs. Guinea-Bissau, and (bottom) Gambia vs. Senegal. The locations sampled in each country for these comparisons were Njabakunda (Gambia), Sedhiou (Senegal), and Antula (Guinea-Bissau). 1228 D. C. Nwakanma et al. selection in limiting gene flow in this pericentromeric out the species range. The application of whole-genome re- region. This also illustrates that mosquitoes typed as M/S sequencing to such studies promises to further increase the heterozygotes in our study generally do not represent F power to map loci under selection and track ongoing changes hybrids between the molecular forms, but are members of in population genetic structure (Cheng et al. 2012). a thoroughly backcrossed and homogenized population, to- gether with S/S homozygotes. Elsewhere in the range of Acknowledgments A. gambiae s.s., major introgression between M- and S-form mosquitoes appears to have been very rare, although it is We thank the staff of our institutes who were involved in strongly suggested by the distribution of insecticide resistance entomological field collections, particularly Sainey Kanteh, genotypes. For example, the kdr mutation conferring resistance Lamin Camara, Alhadji Kaba Sylla, Tolno Gnouma Benoit, to pyrethroids has apparently introgressed from the S form into and Aboubacarr Conteh, and those who assisted in the the M form (Weill et al. 2000; Diabate et al. 2003), and the laboratory, transport, or administration. We appreciate the ace-1(R) mutation has been observed in both M- and S-form encouragement and strong support from institute and de- populations in several West African countries, although it is partmental heads including Tumani Corrah and Moussa unclear in which population this latter mutation originated Dieng Sarr. We acknowledge the expert efforts of members (Djogbenou et al. 2008). of the Biological Samples Platform and Genetic Analysis Reinforcement might occur during the speciation process Platform of the Broad Institute. The genome-wide SNP array by strengthening premating isolation mechanisms, in a situ- was supported by joint funding from several sources, in- ation where postmating isolation is already operating due to cluding Burroughs Wellcome Fund Request 1008238, the reduced fitness of hybrids (Wallace 1889). In areas where M Broad Institute Director’s Fund, Harvard School of Public and S forms have long been in contact, it is possible that Health Department of Immunology and Infectious Diseases they have evolved premating isolation mechanisms to re- under direction of D. F. Wirth, Wellcome Trust Programme duce the frequency of hybridization, such as selective swarm grant 077229/Z/05/Z to F. C. Kafatos and G. K. Christophides, formation (Diabate et al. 2009) or male–female flight-tone and the DeLuca Professorship from Boston College to M.A.T.M. matching (Pennetier et al. 2010). As the S form is more This work was enabled by funding from the United King- widespread, it is possible that S-form populations that have dom Medical Research Council (MRC) and Foundation had no contact with M populations would have had no op- for the National Institutes of Health (FNIH)/Gates Grand portunity to evolve such reinforcement. If M-form popula- Challenges in Global Health to the MRC Unit in The tions are gradually expanding or shifting their range over Gambia. time due to ecological changes and carrying reinforcement mechanisms developed through preceding contact with members of S-form populations, then it may expected that Literature Cited the M form would be generally less susceptible to introgres- Aboagye-Antwi, F., A. Guindo, A. S. Traore, H. Hurd, M. Coulibaly sion. 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Lanzaro, 2001 Distribution of genetic Reproductive isolation in this most important African variation among chromosomal forms of Anopheles gambiae s.s: introgressive hybridization, adaptive inversions, or recent repro- malaria vector species is complex and might not orient ductive isolation? Insect Mol. Biol. 10: 3–7. primarily around the commonly typed M and S forms within Caputo, B., D. Nwakanma, M. Jawara, M. Adiamoh, I. Dia et al., A. gambiae s.s. There is evidence of some genetic substructure 2008 Anopheles gambiae complex along The Gambia river, within each of the forms (Slotman et al. 2007; Weetman et al. with particular reference to the molecular forms of An. gambiae 2010), of ecologically important variation and genomic di- s.s. Malar. J. 7: 182. Caputo,B., F. Santolamazza,J.L.Vicente,D. C. Nwakanma, vergence due to inversion polymorphisms that are not rou- M. 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Lazzaro Interbreeding Between Incipient Species 1231 GENETICS Supporting Information http://www.genetics.org/lookup/suppl/doi:10.1534/genetics.112.148718/-/DC1 Breakdown in the Process of Incipient Speciation in Anopheles gambiae Davis C. Nwakanma, Daniel E. Neafsey, Musa Jawara, Majidah Adiamoh, Emily Lund, Amabelia Rodrigues, Kovana M. Loua, Lassana Konate, Ngayo Sy, Ibrahima Dia, T. Samson Awolola, Marc A. T. Muskavitch, and David J. Conway Copyright © 2013 by the Genetics Society of America DOI: 10.1534/genetics.112.148718 Figure S1 Scatterplot showing correlation of the distribution of values of pooled Contrast ratios for a pool of DNA from 20 mosquitoes, compared with the average values across the 20 DNA samples from the individual mosquitos, with each point corresponding to one feature of the 400K SNP array, for a pilot assay with 20 An gambiae s.s. mosquitos from Mali (Neafsey et al. 2010). 2 SI D. C. Nwakanma et al. Figure S2 Focused profile of differentiation between M and S pools from Guinea Bissau on chromosome 2R (A), 3R (B), and X (C). Black dots indicate Contrast differences between the pools for individual SNP assays. Red dots indicate mean Contrast differences for windows of 50 adjacent SNPs. D. C. Nwakanma et al. 3 SI Figure S3 Test for genomic differentiation between pooled DNA from samples of 20 homozygous M form and 20 homozygous S form Anopheles gambiae s.s. from each of three sites in this study: (A) Njabakunda in The Gambia, (B) Sedhiou in Senegal, (C) Antula in Guinea Bissau, using data from the 66K sub-set of SNPs that had previously shown Hardy-Weinberg equilibrium within molecular forms in Mali (Neafsey et al. 2010). The vertical axis represents divergence measured as the absolute value of mean Contrast difference for stepping windows of 50 assays (this has a modal background of approximately 0.25 which is due to noise rather than a genome-wide divergence signal). The horizontal blue lines indicate the Bonferroni-corrected threshold for statistical significance obtained via bootstrapping. For comparison, panel (D) shows differentiation between similarly pooled DNA from M and S forms in Mali (Neafsey et al. 2010). 4 SI D. C. Nwakanma et al. Figure S4 Comparison of M and S homozygous genotypes with heterozygous genotypes from two study sites: (A) Sedhiou M homozygotes vs. M/S heterozygotes, (B) Sedhiou S homozygotes vs. M/S heterozygotes, (C) Antula M homozygotes vs. M/S heterozygotes, (D) Antula S homozygotes vs. M/S heterozygotes. D. C. Nwakanma et al. 5 SI Table S1 Spearman’s rank correlation coefficients between pairwise pool comparisons of mean Contrast difference in the X pericentromeric region (Mb 15 – 24.2). Table S1 is available for download at http://www.genetics.org/lookup/suppl/doi:10.1534/genetics.112.148718/-/DC1. 6 SI D. C. Nwakanma et al. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Genetics Pubmed Central

Breakdown in the Process of Incipient Speciation in Anopheles gambiae

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Abstract

INVESTIGATION Breakdown in the Process of Incipient Speciation in Anopheles gambiae † ‡ Davis C. Nwakanma,* Daniel E. Neafsey, Musa Jawara,* Majidah Adiamoh,* Emily Lund, § †† ‡‡ §§ Amabelia Rodrigues, Kovana M. Loua,** Lassana Konate, Ngayo Sy, Ibrahima Dia, †,‡,††† ,‡‡‡,1 T. Samson Awolola,*** Marc A. T. Muskavitch, and David J. Conway* † ‡ *Medical Research Council Laboratories, Banjul, The Gambia, Broad Institute, Cambridge, Massachusetts 02467, Harvard School of ** Public Health, Boston, Massachusetts 02115, Bandim Health Project, Bissau, Guinea-Bissau, National Institute of Public Health, †† ‡‡ Conakry, Republic of Guinea, Universite Cheikh Anta Diop, Dakar, Senegal, Service de Lutte Antiparasitaire, Thies, Senegal, §§ ††† *** Institut Pasteur, Dakar, Senegal, Nigerian Institute of Medical Research, Yaba, Lagos, Nigeria, Boston College, Chestnut ‡‡‡ Hill, Massachusetts 02467, and London School of Hygiene and Tropical Medicine, London, WC1E 7HT United Kingdom ABSTRACT Understanding genetic causes and effects of speciation in sympatric populations of sexually reproducing eukaryotes is challenging, controversial, and of practical importance for controlling rapidly evolving pests and pathogens. The major African malaria vector mosquito Anopheles gambiae sensu stricto (s.s.) is considered to contain two incipient species with strong reproductive isolation, hybrids between the M and S molecular forms being very rare. Following recent observations of higher proportions of hybrid forms at a few sites in West Africa, we conducted new surveys of 12 sites in four contiguous countries (The Gambia, Senegal, Guinea- Bissau, and Republic of Guinea). Identification and genotyping of 3499 A. gambiae s.s. revealed high frequencies of M/S hybrid forms at each site, ranging from 5 to 42%, and a large spectrum of inbreeding coefficient values from 0.11 to 0.76, spanning most of the range expected between the alternative extremes of panmixia and assortative mating. Year-round sampling over 2 years at one of the sites in The Gambia showed that M/S hybrid forms had similar relative frequencies throughout periods of marked seasonal variation in mosquito breeding and abundance. Genome-wide scans with an Affymetrix high-density single-nucleotide polymorphism (SNP) micro- array enabled replicate comparisons of pools of different molecular forms, in three separate populations. These showed strong differentiation between M and S forms only in the pericentromeric region of the X chromosome that contains the molecular form- specific marker locus, with only a few other loci showing minor differences. In the X chromosome, the M/S hybrid forms were more differentiated from M than from S forms, supporting a hypothesis of asymmetric introgression and backcrossing. HE major malaria vector mosquito Anopheles gambiae restricted to western parts of Africa, and hybridization between Tsensu stricto (s.s.) exists throughout most of Sub-Saharan them is rare in most areas of sympatry (Della Torre et al. Africa, but there are many polymorphisms including chro- 2005). Scanning of large numbers of polymorphic markers mosomal inversions that appear to be involved in the adap- initiallyindicated themolecular formswereveryhighlydiffer- tation of subpopulations to different environments (Coluzzi entiated at only a small number of discrete regions within the et al. 2002), and molecular forms (M and S) have been iden- two autosomes and the pericentromeric region of the X chro- tified that appear to be reproductively isolated (della Torre mosome, which contains the form-specific markers in the ribo- et al. 2001). The S form is distributed widely throughout the somal (r)RNA genes (Turner et al. 2005). However, a finding A. gambiae species range, whereas the M form is common but that transposable element insertion sites showed marked dif- ferentiation between the forms indicated that reproductive iso- lation might affect a larger portion of the genome (Esnault Copyright © 2013 by the Genetics Society of America doi: 10.1534/genetics.112.148718 et al. 2008). A high-density array to type 400,000 single- Manuscript received September 19, 2012; accepted for publication January 13, 2013 nucleotide polymorphisms (SNPs) has recently enabled more Available freely online through the author-supported open access option. thorough genome-wide analyses, confirming that the molec- Supporting information is available online at http://www.genetics.org/lookup/suppl/ doi:10.1534/genetics.112.148718/-/DC1. ular forms are differentiated at many loci throughout the Corresponding author: Department of Pathogen Molecular Biology, London School of genome in addition to those previously shown (Lawniczak Hygiene and Tropical Medicine, Keppel St., London WC1E 7HT, United Kingdom. E-mail: [email protected] et al. 2010; Neafsey et al. 2010; Reidenbach et al. 2012). Genetics, Vol. 193, 1221–1231 April 2013 1221 It is vital to understand such differentiation and genetic subdivision and its importance in vector evolution, as this complexity can affect malaria control, including resistance to insecticides (Dabire et al. 2009; Djogbenou et al. 2010; Lynd et al. 2010) and susceptibility to malaria parasites and other infections (Rottschaefer et al. 2011; White et al. 2011). Differences in the adult mosquito transcriptome be- tween the M and S molecular forms appear to be minimal (Cassone et al. 2008; Aguilar et al. 2010), but evidence is accumulating that the larval stages are differentially adap- ted to particular features of breeding sites, with the M forms being generally more common in large areas of irrigation for crop cultivation (Diabate et al. 2008; Gimonneau et al. 2011). Studies of mosquito behavior suggest assortative mating may be the means of preventing natural hybridiza- tion between the forms, as most mating swarms consisted of single forms in an area of sympatry in Mali (Diabate et al. 2009), although some mixed swarms were seen in Burkina Faso (Diabate et al. 2006), and very close-range barriers Figure 1 Map of sampling locations for surveys of Anopheles gambiae s.s. molecular forms between 2007 and 2009 in The Gambia, Senegal, to interbreeding may also operate (Pennetier et al. 2010; Guinea-Bissau, and Republic of Guinea. Each location is indicated by Sanford et al. 2011). a yellow circle, and the frequencies of the M form, the S form and M/S Surveys of populations in the extreme west of Africa show hybrid forms sampled at each are indicated by an adjacent pie chart substantial local variation in relative frequencies of A. (sample sizes and exact frequencies are given in Table 1). gambiae s.s. M and S molecular forms (della Torre et al. 2005). For example, the M form predominates in most of employed a pooled hybridization approach for expediency, The Gambia, but ,100 km farther inland in eastern Senegal allowing use of a genotyping array with much higher marker almost all members of the species are S form (Caputo et al. density than that of previous surveys for introgression in West 2008). Of particular interest are areas in which both forms Africa (Marsden et al. 2011; Weetman et al. 2012). Results exist, as the occurrence of natural hybrids may be possible. show extensive hybridization throughout the area and that Elsewhere in Africa, areas of sympatry consistently exhibit mosquitoes with M and S form-specificmarkers are largely very low frequencies of hybrid forms, but a few years ago homogenized throughout the genome, with evidence support- exceptionally high proportions of M/S hybrids were reported ing a hypothesis of asymmetric hybridization and frequent at a few sites near the west coast, with 3% at Dielmo in backcrossing particularly between the M/S hybrid forms and Senegal (Ndiath et al. 2008), 7% at Njabakunda in The Gam- the S forms. bia (Caputo et al. 2008), and 24% at Antula in Guinea-Bissau (Oliveira et al. 2008). Analysis of an insertion polymorphism linked to the form-specific marker on the X chromosome, Materials and Methods as well as an unlinked SNP on chromosome 3, in a subset of Sampling sites for A. gambiae mosquitoes the samples from The Gambia and Guinea-Bissau suggested that there may be introgression between the forms (Caputo Twelve sites were surveyed in four countries (Figure 1). These et al. 2011), and separate studies using broader scans of ge- were chosen to encompass the few areas where unusually high nomic polymorphisms in samples from Guinea-Bissau have frequencies of M/S hybrids had previously been noted and to supported this inference (Marsden et al. 2011; Weetman extend the sampling to the north and south of these, within et al. 2012). 100 km of the west coast of Africa. The northern part of the To thoroughly discover and map the area with high sampling range has Sudan Savannah-type vegetation, with frequencies of hybrids and assess its impact on genomic most annual rainfall of 600–1000 mm normally occurring differentiation, we conducted new surveys of diverse sites in within 4 months (June to September). The south of the sam- the coastal areas of four contiguous countries (The Gambia, pling range has Guinea Savannah-type vegetation including Senegal, Guinea-Bissau, and Republic of Guinea). To in- more woodland, with annual rainfall of up to 2000 mm extend- vestigate the stability of molecular form and hybrid frequen- ing over a longer season (May to October). In all areas, the cies over time throughout different seasons, we conducted majority of A. gambiae s.s. breeding occurs during and at the a longitudinal survey over a 2-year period in the Njabakunda end of the annual rains, and highest densities of adult mosqui- area of The Gambia. We performed high-density genome- toes are found between August and October. The 12 sites are wide scans for differentiation between sympatric M and S numbered and described below (sites 1 and 2 represent Sudan forms, as well as M/S hybrids, at the Njabakunda site and Savannah, sites 3–6 represent northern Guinea Savannah, and at single sites from each of Senegal and Guinea-Bissau. We sites 7–12 represent southern Guinea Savannah). 1222 D. C. Nwakanma et al. Table 1 Proportions of M/S hybrid heterozygote forms and M and S homozygote forms of Anopheles gambiae s.s.in each of 12 sites surveyed from four West African countries Country Site No. mosquitoes M S M/S H F exp IS Gambia Njabakunda 1474 0.164 0.760 0.076 0.322 0.76 Senegal Madina Djikoye 77 0.325 0.350 0.325 0.500 0.35 Senegal Bounkiling 145 0.683 0.138 0.179 0.354 0.49 Senegal Bignona 30 0.767 0.133 0.100 0.295 0.66 Senegal Marsassoum 434 0.270 0.387 0.343 0.493 0.30 Senegal Sedhiou 389 0.653 0.211 0.136 0.402 0.66 Guinea-Bissau Caio 12 0.417 0.167 0.417 0.469 0.11 Guinea-Bissau Antula 464 0.194 0.470 0.336 0.461 0.27 Guinea-Bissau Prabis 91 0.363 0.385 0.253 0.500 0.49 Guinea-Bissau Buba 194 0.907 0.041 0.052 0.130 0.60 Guinea-Bissau Mansoa 152 0.467 0.322 0.210 0.490 0.57 Guinea Boke 37 0.622 0.270 0.108 0.435 0.75 H , expected proportion of M/S heterozygotes under Hardy–Weinberg equilibrium, assuming random mating. There was a significant hetero- exp zygote deficiency (P , 0.005) in each of the populations except for Caio. F inbreeding coefficient (proportion of heterozygotes missing compared IS, to expectations under random mating). The Gambia: Site 1, the Njabakunda village area (13339N, (11489N, 15449W) is a village in a peri-urban area of Bissau 15549W) in the North Bank region of the country (30 km with marsh vegetation and rice cultivation, (site 10) Mansoa west of Farafenni town), was sampled, as previous surveys (1249N, 15199W) is a small town in the north of the coun- (in 2005 and 2006) had shown this area to contain a higher try in a rice cultivation area, and (site 11) Buba (11359N, frequency of M/S form hybrids than elsewhere in The Gam- 1509W) is in the south of the country on the River Rio bia (Caputo et al. 2008). The site is 4 km away from the Grande de Buba near the National Park Contanhez contain- Gambia River on free-draining laterite soil, covered with ing a canopy of tropical rain forest, within which the village open woodland savannah and farmland mainly for cultiva- of Banta Furu was sampled. These sites were sampled in tion of subsistence and cash crops. Four hamlets (Maria August 2009, and Prabis was also sampled in October 2008. Samba Nyado, Sare Illo Buya, Kerr Birom Kardo, and Kerr Sama Kuma) around Njabakunda and within 1 km of each Republic of Guinea: One area in Guinea was sampled in other were selected for longitudinal sampling for a 2-year August 2009, to the south of the border with Guinea-Bissau: period between April 2007 and March 2009. Fortnightly (site 12) near Boke town (10569N, 14189W) four villages (mid-month and end of the month) collections were con- (Korera, Dabaya, Balangdougou, and Bintoumodia) in ter- ducted from June to December covering the rainy season rain of flat plains interspersed with hilly slopes divided by period, and monthly (end of the month) collections were fast-flowing streams, fed by a higher rainfall than at any of done from January to May covering the dry season. the other sites studied here. Collection and identification of mosquitoes Senegal: Five sites were sampled in Senegal. One of these Mosquitoes were sampled from houses or inhabited huts in (site 2), Madina Djikoye (clustered with nearby villages each site, using indoor pyrethrum spray collections (PSC) in Kerr Samba Gueye and Kerr Ousainou Dieng) (13379N, 16189W) is in the Kaolack region immediately to the north most cases for all the sites, with some sampling by indoor of the Gambian border. The other four sites are south of overnight light trap collections (LTC) in addition to PSC in The Gambia in the lower Cassamance region: (site 3) Sed- Guinea-Bissau and Republic of Guinea sites. All mosquitoes hiou (12439N, 1549W) adjacent to the Cassamance River, that were collected were identified morphologically in the (site 4) Bounkiling (1329N, 15429W), (site 5) Bignona field, and A. gambiae sensu lato specimens were stored indi- (12479N, 16149W),and (site6)Marsassoum(12509N, vidually in 1.5-ml polyethylene tubes with desiccant or in 15589W). The sites have free-draining laterite sand or al- Carnoy’s solution if they were semigravid, for subsequent luvial soil and are covered with open woodland savannah DNA typing of species and molecular forms. DNA was ex- or farmland for groundnut and cereal cultivation. These tracted from individual mosquito specimens, using the Corbett sites were sampled in August 2009, except for Sedhiou, Robotics X-Tractor Gene automated DNA extraction platform which was sampled in October 2008. (QIAGEN, Crawley, UK) according to the manufacturer’s protocol for tissue extraction. Whole mosquito material was Guinea-Bissau: Five sites were sampled in Guinea-Bissau: extracted, except for a few legs of each specimen retained for (site 7) Caio village (11559N, 16129W) is in a coastal area confirmatory PCR testing in case the initial result indicated of rice and cashew plantations mixed with woodland a hybrid molecular form. Each of the three species of the A. with traditional ritual significance, (site 8) Antula (11539N, gambiae complex that exist in West Africa (A. gambiae s.s., A. 15359W) is a village east of the capital Bissau, (site 9) Prabis arabiensis,and A. melas) were discriminated in the laboratory Interbreeding Between Incipient Species 1223 Figure 2 Numbers of molecular forms M and S and M/S hybrid forms sampled in each month from a total of 1474 Anopheles gambiae s.s. individuals collected in the Njabakunda area of The Gambia over a 2-year period from April 2007 until March 2009. by species-specificPCR of sequenceswithinthe rRNA gene individual hybridizations and pooled hybridizations for pools locus on the X chromosome (Scott et al. 1993), and A. gambiae of 20 individuals (Pearson’s r =0.96, Supporting Informa- s.s. mosquitoes were differentiated into molecular forms M tion, Figure S1), leading to the repetition of that assay design and S (and M/S hybrids) by form-specific restriction enzyme here. Following Robust Multi-array Average (RMA) back- digestion of the amplified fragment (Fanello et al. 2002). ground correction and quantile normalization using Affyme- trix Power Tools, allelic balance at assayed loci within the Genome-wide scans for differentiation between pools pools was inferred using the Contrast statistic generated by of A. gambiae s.s. mosquitoes BRLMM-P, using a K value of 1 (Rabbee and Speed 2007). A custom Affymetrix oligonucleotide array for genome-wide Such plots result in a modal background mean Contrast dif- typing of 400,071 A. gambiae s.s. SNPs (Neafsey et al. 2010), ference of 0.25 rather than 0, which is the result of noise in termed the 400K array, was used for population genotyping. the signal rather than a genome-wide divergence signal. Plots DNA hybridization was performed with separate pooled of local divergence along the chromosomes were generated samples of 20 homozygous M-form and 20 homozygous using a stepping window approach, by calculating the mean S-form A. gambiae s.s. mosquitoes from each of three sites of the absolute value of the arithmetic difference in Contrast sampled (Njabakunda in The Gambia, Sedhiou in Senegal, for 50 adjacent assays. The mean physical distance spanned and Antula in Guinea-Bissau). The Njabakunda site was the by 50 adjacent assays was 28.2 kb (range = 9.1–280 kb, primary one selected for this genome-wide SNP analysis, as standard deviation = 16.6 kb). Thresholds for statistical sig- we studied this site intensively. Therefore, it had the largest nificance (a = 0 0.05 after Bonferroni correction) for these sample size and most accurate estimation of frequencies. We windowed analyses were calculated using a bootstrapping added the other two sites to include one from Senegal and approach, in which mean Contrast difference was calculated one from Guinea-Bissau, each of which had large sample for each pairwise comparison for 50 SNPs that were sampled sizes with higher proportions of hybrids. In addition, a pool randomly from the genome a total of 10 million times. of DNA from 20 M/S hybrid mosquitoes was tested and compared with the homozygous samples, for each of these Results three sites. Each of the 180 individual mosquitoes that con- Quantifying proportions of molecular forms and hybrids tributed to the pooled arrays (20 M form, 20 S form, and at different sites 20 M/S forms, selected from each of the three sites noted) was also genotyped for the SINE200x6.1 transposon insertion Molecular form typing was performed on a total of 3499 A. site polymorphism that is linked to the molecular form marker gambiae s.s. mosquitoes sampled from the 12 sites surveyed in on the X chromosome (Santolamazza et al. 2011). Affymetrix the four countries (Senegal, The Gambia, Guinea-Bissau, and oligonucleotide array hybridization was performed following Republic of Guinea) (Figure 1). In each site, M and S molecular methods already described (Neafsey et al. 2010), as previous forms coexisted sympatrically and substantial proportions of analysis revealed a very high correlation between aggregate M/S hybrid forms were found (from 5 to 42%) (Table 1). The 1224 D. C. Nwakanma et al. Figure 3 Test for genomic differentiation between pooled DNA from samples of 20 homozygous M-form and 20 homozygous S-form Anopheles gambiae s.s. from each of three sites in this study: (A) Njabakunda in The Gambia, (B) Sedhiou in Senegal, and (C) Antula in Guinea-Bissau. The vertical axis represents divergence measured as the absolute value of mean Contrast difference for stepping windows of 50 assays (this has a modal background of 0.25 that is due to noise rather than a genome-wide divergence signal), using data from all SNPs on the 400K genome-wide array. The horizontal blue lines indicate the Bonferroni- corrected threshold for statistical significance obtained via bootstrapping. Only the X-chromosomal pericentromeric region that contains the M and S molecular form markers exhibits differentiation. For comparison, D shows differentiation between similarly pooled DNA from M and S forms in Mali (Neafsey et al. 2010). estimated inbreeding coefficients for this rRNA gene locus, portions of M/S hybrids were less than expected under Hardy– which has previously been considered a putative speciation Weinberg equilibrium with complete panmixia, but in some marker, showed a wide spectrum of values (from 0.11 to 0.76) cases not much less. (Table 1). Seven of the sites each had samples of .100 mos- Analysis of proportions of molecular forms and hybrids quitoes analyzed, allowing highly accurate estimations of at one site throughout a 2-year period frequencies, and we note that these also show a wide range of M/S hybrid proportions (from 5 to 34%) and estimated To investigate the stability of frequencies over time, we sampled inbreeding coefficients (from 0.27 to 0.76). Thus, the pro- the Njabakunda site in The Gambia throughout a 2-year period Interbreeding Between Incipient Species 1225 Table 2 Number of 50-SNP windows exhibiting significant (P , 0.05) mean contrast differences according to pairwise pool comparison and chromosomal location 2R: 2L: 3R: 3L: X: Chi-square P-value: Total windows n = 2256 n = 1846 n = 1692 n = 1374 n = 829 X vs. autosomes (compared with Mali) Gambia M vs. S 3 2 2 1 42 1.70 · 10 Senegal M vs. S 9 3 1 0 98 7.17 · 10 Guinea-Bissau M vs. S 0 0 0 0 58 7.20 · 10 Mali M vs. S 20 30 21 11 112 Comparator Gambia M vs. M/S 5 3 1 0 37 1.83 · 10 Gambia S vs. M/S 0 1 0 0 0 Not applicable (annual periods extending from April to the following March to other African populations (Figure 3D shows published data capture full seasonal dynamics due to rainfall), yielding 1474 from Mali for comparison). A summary of the number of typed mosquitoes from this site (1048 in the first annual period nonoverlapping windows of 50 adjacent SNPs from each and 426 in the second). The relative frequencies of M/S hybrid chromosomal arm exhibiting significant differentiation in forms were stable in both years, accounting for 8.3% in the first each pairwise comparison is presented in Table 2. M vs. S and 5.6% in the second year (chi-square test, P = 0.07) (Figure comparisons for each of the three separate sites exhibit a sig- 2). During each year, S-form mosquito numbers peaked early in nificantly higher ratio of divergent windows on the X chro- the rainy season, and the peak of the less abundant M form mosome compared to the autosomes, relative to the more occurred later in the season, but M/S hybrid forms were canonical interform divergence profile exhibited by the seen throughout. In 2007, M/S forms had seasonally Mali comparison (chi-square tests on the ratio at each stable frequencies of 8.6% (32/374) in April–July and of the sites compared with Mali: P = 1.7 · 10 for the 8.1% (50/620) in August–October (chi-square test, P = Gambian site and P , 10 for the sites in Senegal and 0.78), while proportions of M forms increased from 1.9% Guinea-Bissau) (Table 2). (Table S1 includes the Contrast (7/374) to 22.6% (140/620) during those respective peri- Difference datafile). ods (chi-square test, P , 10 ); in 2008, M/S forms had To test whether minor signals of differentiation may have frequencies of 4.9% (9/184) in April–July and 5.9% (12/ been being obscured by our windowed analysis approach, 205) in August–October (chi-square test, P =0.67),while we examined the region between 28 and 42 Mb on chro- proportions of M forms increased from 8.1% (15/184) to mosome 2R in closer detail, at the level of individual SNP 36.6% (75/205) in those periods (chi-square test, P = assays, for the comparison between M- and S-form pools in 10 ). Guinea-Bissau (Figure S2). This region was focused on as it contains loci that showed differentiation between the M and Genome-wide scans for differentiation between S forms in a recent study, using a panel of several hundred molecular forms within sympatric samples SNPs with a sample from Guinea-Bissau (Weetman et al. To identify whether marked genomic differentiation be- 2012). Although some individual SNP assays in the region tween M and S forms could be seen, we randomly selected showed high Contrast differences between the pools, the 20 homozygous M-form and 20 homozygous S-form Contrast distributions were similar to those observed in mosquitoes from the Njabakunda population for a pooled- a control region of chromosome 3R (19–32 Mb) and much population genome-wide scan of single-nucleotide polymor- lower than those observed in the highly differentiated X phism differentiation, using the custom Affymetrix 400K centromeric region (Figure S2). To test whether signals of array. Remarkably, with the clear exception of the X chro- differentiation might have been obscured by noise due to mosome pericentromeric region that contains the specific poorly performing SNP genotyping on the array, we re- polymorphism used to stratify the molecular forms, there peated the genome-wide analysis with a subset of 65,974 were no loci that exhibited strong differentiation between SNPs that had previously been validated and shown to be in the M and S molecular forms (Figure 3A). To replicate the Hardy–Weinberg equilibrium for individual molecular forms test, genome-wide comparison between M and S forms in Mali (Neafsey et al. 2010). This confirmed that, outside of was conducted for pools of 20 M-form and 20 S-form the pericentromeric X-chromosomal region, there was no ma- mosquitoes from each of two other sites that also exhibit jor differentiation between the forms in the populations stud- high frequencies of M/S hybrids, at Sedhiou in Senegal ied here, in contrast to the interform differentiation previously seen in Mali (Figure S3). We cannot fully reconcile this obser- (Figure 3B) and Antula in Guinea-Bissau (Figure 3C). Both vation with reports of very narrow regions of differentiation of these sites similarly showed a lack of substantial differen- tiation except in the X-chromosomal pericentromeric region. on 2R in another population sample from Guinea-Bissau This contrasts strongly with the observation of differentia- (Weetman et al. 2012), as differences in findings between tion at pericentromeric autosomal loci and elsewhere the studies may result from fine differences in spatial or tem- throughout the genome of M and S forms sampled from poral sampling or in the sensitivity of the respective assays. 1226 D. C. Nwakanma et al. resultsthatwereconcordantwiththe molecularformtyp- ing in 159 (88%) of the individuals. While there was 98% (59/60) concordance for the M form and 100% (60/60) for the S form, confirming appropriate sample selection and robust comparison of the forms here, the lower con- cordance of 67% (40/60) observed for the M/S forms con- firms that many of these are not F hybrids, but that backcrossing must have occurred over multiple genera- tions. The profile of windows with significant differentia- tion in M vs. M/S comparisons is highly similar among the three countries sampled (Figure 5). Across the pairs of countries, M vs. M/S comparisons exhibit Spearman’s rank correlations ranging between 0.77 and 0.84, whereas S vs. M/S comparisons have Spearman’s rank correlations of only 0.15–0.42 between the same pairs (Table S1). Discussion It is widely considered that the major malaria vector A. gambiae s.s. is undergoing a process of speciation into at least two reproductive units defined as the M and S molec- Figure 4 Genome-wide comparison of an M/S pool of 20 hybrid form mosquitoes with M and S homozygous pools from Njabakunda in The ular forms (della Torre et al. 2001). Although the divergence Gambia, using data from all SNPs on the 400K genome-wide array: (top) of these forms is likely proceeding in many parts of west and M homozygotes vs. M/S hybrid forms; (bottom) S homozygotes vs. M/S central Africa (Reidenbach et al. 2012), our results and hybrid forms. The more complete lack of differentiation between S and those of other recent surveys of populations in the extreme M/S suggests asymmetric gene flow between the forms or selection to west of Africa (Caputo et al. 2011; Marsden et al. 2011; maintain the residual differentiation in the M form. (Replicate compari- sons between the pooled hybrid forms and each of the major forms are Oliveira et al. 2008; Weetman et al. 2012) indicate that shown for two separate populations in Figure S4.) hybridization is common. A contiguous region is now out- lined, covering ecologically diverse sites sampled in a contig- uous area within 100 km of the West African coast within Asymmetric minimal differentiation between hybrid four countries in a zone of ,400 km from north to south, forms and homozygous molecular forms where frequencies of M/S hybrid forms are consistently We also performed pooled array hybridizations, using 20 much higher than elsewhere, and there is minimal interform mosquitoes genotyped as M/S heterozygote “hybrid forms” genomic differentiation outside of the pericentromeric re- from each site and compared these with the M and S homo- gion of the X chromosome. This contrasts significantly with zygote pools. Results for the Gambian population sample the results obtained from populations farther east, using the (Figure 4, Table 2) show no divergence in the X pericentro- same methods (Reidenbach et al. 2012), and is important meric region between S and M/S, whereas some divergence for understanding evolution and implementing control of remains between M and M/S. The degree of differentiation this malaria vector and for investigation of the processes observed in the X pericentromeric region (megabases 15– of speciation. 24.2) is significantly higher in the M vs. M/S comparison A wide range of proportions of M/S hybrid forms was relative to the S vs. M/S comparison (Wilcoxon’s sign-rank detected within both Guinea-Bissau and Senegal, with the test, V = 25,996, P = 2.2 · 10 ). Replication of these Gambian site in the north and the Guinean site in the south comparisons for samples from Sedhiou in Senegal and both showing lower proportions than many of the interven- Antula in Guinea-Bissau showed similar results (Figure ing sites. It appears likely that the area we have surveyed, S4), except that the S and M/S pools showed some detect- extending across these four countries, probably covers most able X pericentromeric differentiation in Senegal (less of the current geographical range of the A. gambiae s.s pop- than observed for the comparison of M and M/S). This ulations that exhibit extensive hybridization. Surveys imme- suggests that although the forms are now highly homoge- diately to the south in Guinea have shown mostly S-form nized, the speed of the process has been asymmetric, with mosquitoes near the coast, and both M and S forms, with partial isolation or a lower recombination rate allowing a very low frequency of hybrids, further inland (della Torre maintenance of greater X chromosome pericentromeric di- et al. 2005; Vezenegho et al. 2009; Carnevale et al. 2010). In vergence in the M form. Genotyping of the SINE200 trans- northern Senegal, A. gambiae s.s. is less common than the poson insertion site polymorphism in the 180 mosquitoes related vector species A. arabiensis and exists mostly as the contributing to the pooled arrays (20M form,20S form, M form (della Torre et al. 2005; Dia et al. 2008), while and 20 M/S forms, in each of the three selected sites) gave surveys in the eastern part of Senegal have shown the S Interbreeding Between Incipient Species 1227 form to be more abundant than the M form, with hybrid forms very rarely detected (della Torre et al. 2005; Caputo et al. 2008), as has been observed in Mali and further east- ward (della Torre et al. 2005; Diabate et al. 2009; Aboagye- Antwi et al. 2010). It is possible that our results reflect consequences of the reversal of a previous process that promotes speciation, with the genomic integrity of one or both of the molecular forms now being substantially compromised by introgression within these western populations. It was previously sugges- ted that areas might exist within which there is extensive gene flow between the molecular forms of A. gambiae s.s., while reproductive barriers are maintained at other sites (Black and Lanzaro 2001; Lehmann et al. 2003). A more recent view, also supported by pooled array hybridizations, is that sporadic bouts of hybridization mediated by changing ecological conditions or a very low background rate of gene flow between M and S forms could be responsible for the relative homogeneity of M and S genomes outside of the “speciation islands” (Reidenbach et al. 2012). Such pro- cesses would not be expected to disrupt the divergence at well-established pericentromeric islands of speciation, however. Our results enable sites to be specifically selected for population genomic studies to investigate mechanisms of reproductive isolation. Studies of male mating swarm composition and behavior will be important to conduct in these areas, to identify whether there are proximal determi- nants of hybridization (Diabate et al. 2006, 2009). Further sampling and measurement of multiple ecological parame- ters at different times of the year are also needed to char- acterize these and additional sites more fully, which could enable exploration of correlations between environmental variables, population genetic structure, and reproductive isolation. If the M and S forms were undergoing genome-wide reciprocal introgression, it is expected that the pericentro- meric divergence on the X chromosome should be gradually eliminated in both forms by recombination. The observation that the X pericentromeric region remains relatively distinct in M-form populations in this area, as also reported recently by others sampling in Guinea-Bissau (Marsden et al. 2011), supports a hypothesis that partial reproductive isolation or selective pressure preserves a narrow genomic island linked to the M-form-specific marker (Caputo et al. 2011; Marsden et al. 2011; Weetman et al. 2012). A high degree of similar- ity in the specific pericentromeric divergence profile among sites (Figure 5, Table S2) further indicates the likely role of Figure 5 Comparison of M vs. M/S divergence among geographic sites in the X pericentromeric region (megabases 15–24.2). Each point represents a 50-SNP window. The profile of differentiation is maintained in a highly similar manner in all pairwise comparisons of geographic sites: (top) Gambia vs. Guinea-Bissau, (middle) Senegal vs. Guinea-Bissau, and (bottom) Gambia vs. Senegal. The locations sampled in each country for these comparisons were Njabakunda (Gambia), Sedhiou (Senegal), and Antula (Guinea-Bissau). 1228 D. C. Nwakanma et al. selection in limiting gene flow in this pericentromeric out the species range. The application of whole-genome re- region. This also illustrates that mosquitoes typed as M/S sequencing to such studies promises to further increase the heterozygotes in our study generally do not represent F power to map loci under selection and track ongoing changes hybrids between the molecular forms, but are members of in population genetic structure (Cheng et al. 2012). a thoroughly backcrossed and homogenized population, to- gether with S/S homozygotes. Elsewhere in the range of Acknowledgments A. gambiae s.s., major introgression between M- and S-form mosquitoes appears to have been very rare, although it is We thank the staff of our institutes who were involved in strongly suggested by the distribution of insecticide resistance entomological field collections, particularly Sainey Kanteh, genotypes. For example, the kdr mutation conferring resistance Lamin Camara, Alhadji Kaba Sylla, Tolno Gnouma Benoit, to pyrethroids has apparently introgressed from the S form into and Aboubacarr Conteh, and those who assisted in the the M form (Weill et al. 2000; Diabate et al. 2003), and the laboratory, transport, or administration. We appreciate the ace-1(R) mutation has been observed in both M- and S-form encouragement and strong support from institute and de- populations in several West African countries, although it is partmental heads including Tumani Corrah and Moussa unclear in which population this latter mutation originated Dieng Sarr. We acknowledge the expert efforts of members (Djogbenou et al. 2008). of the Biological Samples Platform and Genetic Analysis Reinforcement might occur during the speciation process Platform of the Broad Institute. The genome-wide SNP array by strengthening premating isolation mechanisms, in a situ- was supported by joint funding from several sources, in- ation where postmating isolation is already operating due to cluding Burroughs Wellcome Fund Request 1008238, the reduced fitness of hybrids (Wallace 1889). In areas where M Broad Institute Director’s Fund, Harvard School of Public and S forms have long been in contact, it is possible that Health Department of Immunology and Infectious Diseases they have evolved premating isolation mechanisms to re- under direction of D. F. Wirth, Wellcome Trust Programme duce the frequency of hybridization, such as selective swarm grant 077229/Z/05/Z to F. C. Kafatos and G. K. Christophides, formation (Diabate et al. 2009) or male–female flight-tone and the DeLuca Professorship from Boston College to M.A.T.M. matching (Pennetier et al. 2010). As the S form is more This work was enabled by funding from the United King- widespread, it is possible that S-form populations that have dom Medical Research Council (MRC) and Foundation had no contact with M populations would have had no op- for the National Institutes of Health (FNIH)/Gates Grand portunity to evolve such reinforcement. If M-form popula- Challenges in Global Health to the MRC Unit in The tions are gradually expanding or shifting their range over Gambia. time due to ecological changes and carrying reinforcement mechanisms developed through preceding contact with members of S-form populations, then it may expected that Literature Cited the M form would be generally less susceptible to introgres- Aboagye-Antwi, F., A. Guindo, A. S. Traore, H. Hurd, M. Coulibaly sion. The demographic and geographic range history of the et al., 2010 Hydric stress-dependent effects of Plasmodium fal- forms is currently unclear, however. Behavioral mechanisms, ciparum infection on the survival of wild-caught Anopheles gam- such as the relative importance of male or female mate biae female mosquitoes. Malar. J. 9: 243. Aguilar, R., F. Simard, C. Kamdem, T. Shields, G. E. Glass et al., choice in determining prezygotic isolation, could also play 2010 Genome-wide analysis of transcriptomic divergence be- an important role. Further investigation of the ecology and tween laboratory colony and field Anopheles gambiae mosquitoes range-wide demographic history of these forms may be re- of the M and S molecular forms. Insect Mol. Biol. 19: 695–705. quired to understand the nature of the observed introgression. Black, W. C., and G. C. 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Lazzaro Interbreeding Between Incipient Species 1231 GENETICS Supporting Information http://www.genetics.org/lookup/suppl/doi:10.1534/genetics.112.148718/-/DC1 Breakdown in the Process of Incipient Speciation in Anopheles gambiae Davis C. Nwakanma, Daniel E. Neafsey, Musa Jawara, Majidah Adiamoh, Emily Lund, Amabelia Rodrigues, Kovana M. Loua, Lassana Konate, Ngayo Sy, Ibrahima Dia, T. Samson Awolola, Marc A. T. Muskavitch, and David J. Conway Copyright © 2013 by the Genetics Society of America DOI: 10.1534/genetics.112.148718 Figure S1 Scatterplot showing correlation of the distribution of values of pooled Contrast ratios for a pool of DNA from 20 mosquitoes, compared with the average values across the 20 DNA samples from the individual mosquitos, with each point corresponding to one feature of the 400K SNP array, for a pilot assay with 20 An gambiae s.s. mosquitos from Mali (Neafsey et al. 2010). 2 SI D. C. Nwakanma et al. Figure S2 Focused profile of differentiation between M and S pools from Guinea Bissau on chromosome 2R (A), 3R (B), and X (C). Black dots indicate Contrast differences between the pools for individual SNP assays. Red dots indicate mean Contrast differences for windows of 50 adjacent SNPs. D. C. Nwakanma et al. 3 SI Figure S3 Test for genomic differentiation between pooled DNA from samples of 20 homozygous M form and 20 homozygous S form Anopheles gambiae s.s. from each of three sites in this study: (A) Njabakunda in The Gambia, (B) Sedhiou in Senegal, (C) Antula in Guinea Bissau, using data from the 66K sub-set of SNPs that had previously shown Hardy-Weinberg equilibrium within molecular forms in Mali (Neafsey et al. 2010). The vertical axis represents divergence measured as the absolute value of mean Contrast difference for stepping windows of 50 assays (this has a modal background of approximately 0.25 which is due to noise rather than a genome-wide divergence signal). The horizontal blue lines indicate the Bonferroni-corrected threshold for statistical significance obtained via bootstrapping. For comparison, panel (D) shows differentiation between similarly pooled DNA from M and S forms in Mali (Neafsey et al. 2010). 4 SI D. C. Nwakanma et al. Figure S4 Comparison of M and S homozygous genotypes with heterozygous genotypes from two study sites: (A) Sedhiou M homozygotes vs. M/S heterozygotes, (B) Sedhiou S homozygotes vs. M/S heterozygotes, (C) Antula M homozygotes vs. M/S heterozygotes, (D) Antula S homozygotes vs. M/S heterozygotes. D. C. Nwakanma et al. 5 SI Table S1 Spearman’s rank correlation coefficients between pairwise pool comparisons of mean Contrast difference in the X pericentromeric region (Mb 15 – 24.2). Table S1 is available for download at http://www.genetics.org/lookup/suppl/doi:10.1534/genetics.112.148718/-/DC1. 6 SI D. C. Nwakanma et al.

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Genetics – Pubmed Central

Published: Apr 1, 2013

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