FIG. 1.— Classical scenarios for the evolution of a new function through duplication. The different scenarios for the evolution of a new function through.

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FIG. 1.— Classical scenarios for the evolution of a new function through duplication. The different scenarios for the evolution of a new function through duplication proposed in the literature are illustrated. The new function is represented in gray. Short-term and long-term events are distinguished and the evolutionary force implied is indicated (in bold). Note that the case of subfunctionalization can be only considered to lead to the evolution of new functions as far as further improvement of at least one of the subfunction takes place. From: Independent Duplications of the Acetylcholinesterase Gene Conferring Insecticide Resistance in the Mosquito Culex pipiens Mol Biol Evol. 2007;24(4):1056-1067. doi:10.1093/molbev/msm025 Mol Biol Evol | © The Author 2007. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

FIG. 2. — Duplicated haplotype detection protocol FIG. 2.— Duplicated haplotype detection protocol. Females of the strain tested with an [RS] phenotype were crossed with SLAB males (genotype (S/S)). Their progenies were then selected individually, using propoxur insecticide at a concentration killing only susceptible individuals. If no mortality was detected in the progeny, the female was identified as bearing a duplicated ace-1 gene and was thus analyzed. If some or all of the progeny died following exposure to insecticide, the corresponding female was eliminated. From: Independent Duplications of the Acetylcholinesterase Gene Conferring Insecticide Resistance in the Mosquito Culex pipiens Mol Biol Evol. 2007;24(4):1056-1067. doi:10.1093/molbev/msm025 Mol Biol Evol | © The Author 2007. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

FIG. 3.— Diversity of the ace-1 copies in Culex pipiens pipiens samples. Variability of the different resistant D<sub>i</sub>(R) and susceptible D<sub>i</sub>(S) copies of each duplicated haplotype ace-1<sup>Di</sup> and the single copy alleles R and S found in the Ganges sample (G) are presented (Jukes–Cantor distance, ClustalW (1.83) software [Thomopson et al. 1994]), 1,000 bootstraps. The sequenced of ace-1 considered is a part of the exon 3, for each copy. The G119S mutation, selected for resistance to OP, has been removed to consider only the neutral variation. Bootstraps are presented in percentage. From: Independent Duplications of the Acetylcholinesterase Gene Conferring Insecticide Resistance in the Mosquito Culex pipiens Mol Biol Evol. 2007;24(4):1056-1067. doi:10.1093/molbev/msm025 Mol Biol Evol | © The Author 2007. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

FIG. 4.— Diversity of the ace-1 copies in Culex pipiens quinquefasciatus samples. Variability of the different resistant D<sub>i</sub>(R) and susceptible D<sub>i</sub>(S) copies of each duplicated haplotype ace-1<sup>Di</sup> and the single copy alleles R and S found in the Ducos (Du) and Palawan (Pa) samples are presented (Jukes–Cantor distance, ClustalW (1.83) software [Thomopson et al. 1994]), 1,000 bootstraps). The sequenced of ace-1 considered encompassed the intron 2 and the exon 3, for each copy. The G119S mutation, selected for resistance to OP, has been removed to consider only the neutral variation. Bootstraps are presented in percentage. Considering only the part of the exon 3 available for every individual sequenced, this figure basically connects to figure 3 in a straightforward way, with 3 diagnostic mutations separating the sequences from the 2 subspecies. From: Independent Duplications of the Acetylcholinesterase Gene Conferring Insecticide Resistance in the Mosquito Culex pipiens Mol Biol Evol. 2007;24(4):1056-1067. doi:10.1093/molbev/msm025 Mol Biol Evol | © The Author 2007. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

FIG. 5. — Origin of ace-1 duplications FIG. 5.— Origin of ace-1 duplications. Sensus stricto duplications (Type A event): scenario 3a unequal crossing-over or replication slippage in a resistant homozygote followed by reversion to susceptibility or recombination with a susceptible allele; scenario 3b unequal crossing-over or replication slippage in a susceptible homozygote followed by mutation toward resistance or recombination with a resistance allele; scenario 4 unequal crossing-over in an heterozygote individual. Examples of secondary recombination events that could modify the haplotype sequence are illustrated. Type B event: recombination in an individual carrying both a duplicated haplotype and a single copy allele, either a R allele (a) or a S allele (b). Type C event: crossing-over in an individual carrying 2 different duplicated haplotypes. Similar sequences are illustrated with the same color. Resistant copies are indicated by a bar representing the G119S mutation. From: Independent Duplications of the Acetylcholinesterase Gene Conferring Insecticide Resistance in the Mosquito Culex pipiens Mol Biol Evol. 2007;24(4):1056-1067. doi:10.1093/molbev/msm025 Mol Biol Evol | © The Author 2007. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org