The chromosome-scale assembly of the Notopterygium incisum genome provides insight into the structural diversity of coumarins.

Coumarins, derived from the phenylpropanoid pathway, represent one of the primary metabolites found in angiosperms. The alignment of the tetrahydropyran (THP) and tetrahydrofuran (THF) rings with the lactone structure results in the formation of at least four types of complex coumarins. However, the mechanisms underlying the structural diversity of coumarin remain poorly understood. Here, we report the chromosome-level genome assembly of Notopterygium incisum, spanning 1.64 Gb, with a contig N50 value of 22.7 Mb and 60,021 annotated protein-coding genes. Additionally, we identified the key enzymes responsible for shaping the structural diversity of coumarins, including two p-coumaroyl CoA 2'-hydroxylases crucial for simple coumarins basic skeleton architecture, two UbiA prenyltransferases responsible for angular or linear coumarins biosynthesis, and five CYP736 cyclases involved in THP and THF ring formation. Notably, two bifunctional enzymes capable of catalyzing both demethylsuberosin and osthenol were identified for the first time. Evolutionary analysis implies that tandem and ectopic duplications of the CYP736 subfamily, specifically arising in the Apiaceae, contributed to the structural diversity of coumarins in N. incisum. Conclusively, this study proposes a parallel evolution scenario for the complex coumarin biosynthetic pathway among different angiosperms and provides essential synthetic biology elements for the heterologous industrial production of coumarins.

Parallel evolution of methyltransferases leads to vobasine biosynthesis in Tabernaemontana elegans and Catharanthus roseus.

Monoterpenoid indole alkaloids (MIA) are one of the largest and most complex alkaloid class in nature, boasting many clinically significant drugs such as anticancer vinblastine and antiarrhythmic ajmaline. Many MIAs undergo nitrogen N-methylation, altering their reactivity and affinity to the biological targets through a straightforward reaction. Remarkably, all known MIA N-methyltransferases (NMT) originate from the neofunctionalization of ancestral γ-tocopherol C-methyltransferases (γTMTs), a phenomenon seemingly unique to the Apocynaceae family. In this study, we unveil and characterize a new γTMT-like enzyme from the plant Tabernaemontana elegans (toad tree): perivine Nß-methyltransferase (TePeNMT). TePeNMT and other homologs form a distinct clade in our phylogenetic study, setting them apart from other γTMTs and γTMT-like NMTs discovered to date. Enzyme kinetic experiments and enzyme homology modeling studies reveal the significant differences in enzyme active sites between TePeNMT and CrPeNMT, a previously characterized perivine Nß-methyltransferase from Catharanthus roseus (Madagascar periwinkle). Collectively, our findings suggest that parallel evolution of ancestral γTMTs may be responsible for the occurrence of perivine N-methylation in T. elegans and C. roseus.

Comparative histology of abscission zones reveals the extent of convergence and divergence in seed shattering in weedy and cultivated rice.

The modification of seed shattering has been a recurring theme in rice evolution. The wild ancestor of cultivated rice disperses its seeds, but reduced shattering was selected during multiple domestication events to facilitate harvesting. Conversely, selection for increased shattering occurred during the evolution of weedy rice, a weed invading cultivated rice fields that has originated multiple times from domesticated ancestors. Shattering requires formation of a tissue known as the abscission zone (AZ), but how the AZ has been modified throughout rice evolution is unclear. We quantitatively characterized the AZ characteristics of relative length, discontinuity, and intensity in 86 cultivated and weedy rice accessions. We reconstructed AZ evolutionary trajectories and determined the degree of convergence among different cultivated varieties and among independent weedy rice populations. AZ relative length emerged as the best feature to distinguish high and low shattering rice. Cultivated varieties differed in average AZ morphology, revealing lack of convergence in how shattering reduction was achieved during domestication. In contrast, weedy rice populations typically converged on complete AZs, irrespective of origin. By examining AZ population-level morphology, our study reveals its evolutionary plasticity, and suggests that the genetic potential to modify the ecologically and agronomically important trait of shattering is plentiful in rice lineages.

Heterogeneous genomic architecture of skeletal armour traits in sticklebacks.

Whether populations adapt to similar selection pressures using the same underlying genetic variants depends on population history and the distribution of standing genetic variation at the metapopulation level. Studies of sticklebacks provide a case in point: when colonizing and adapting to freshwater habitats, three-spined sticklebacks (Gasterosteus aculeatus) with high gene flow tend to fix the same adaptive alleles in the same major loci, whereas nine-spined sticklebacks (Pungitius pungitius) with limited gene flow tend to utilize a more heterogeneous set of loci. In accordance with this, we report results of quantitative trait locus (QTL) analyses using a backcross design showing that lateral plate number variation in the western European nine-spined sticklebacks mapped to 3 moderate-effect QTL, contrary to the major-effect QTL in three-spined sticklebacks and different from the 4 QTL previously identified in the eastern European nine-spined sticklebacks. Furthermore, several QTL were identified associated with variation in lateral plate size, and 3 moderate-effect QTL with body size. Together, these findings indicate more heterogenous and polygenic genetic underpinnings of skeletal armour variation in nine-spined than three-spined sticklebacks, indicating limited genetic parallelism underlying armour trait evolution in the family Gasterostidae.

Parallel Evolution in Mosquito Vectors-A Duplicated Esterase Locus is Associated With Resistance to Pirimiphos-methyl in Anopheles gambiae.

The primary control methods for the African malaria mosquito, Anopheles gambiae, are based on insecticidal interventions. Emerging resistance to these compounds is therefore of major concern to malaria control programs. The organophosphate (OP), pirimiphos-methyl, is a relatively new chemical in the vector control armory but is now widely used in indoor-residual spray campaigns. While generally effective, phenotypic resistance has developed in some areas in malaria vectors. Here, we used a population genomic approach to identify novel mechanisms of resistance to pirimiphos-methyl in A. gambiae s.l mosquitoes. In multiple populations, we found large and repeated signals of selection at a locus containing a cluster of detoxification enzymes, some of whose orthologs are known to confer resistance to OPs in Culex pipiens. Close examination revealed a pair of alpha-esterases, Coeae1f and Coeae2f, and a complex and diverse pattern of haplotypes under selection in A. gambiae, A. coluzzii and A. arabiensis. As in C. pipiens, copy number variants have arisen at this locus. We used diplotype clustering to examine whether these signals arise from parallel evolution or adaptive introgression. Using whole-genome sequenced phenotyped samples, we found that in West Africa, a copy number variant in A. gambiae is associated with resistance to pirimiphos-methyl. Overall, we demonstrate a striking example of contemporary parallel evolution which has important implications for malaria control programs.

Alternative splicing in parallel evolution and the evolutionary potential in sticklebacks.

Repeatability of adaptation to similar environments provides opportunity to evaluate the predictability of natural selection. While many studies have investigated gene expression differences between populations adapted to contrasting environments, the role of post-transcriptional processes such as alternative splicing has rarely been evaluated in the context of parallel adaptation. To address the aforementioned knowledge gap, we reanalysed transcriptomic data from three pairs of threespine stickleback (Gasterosteus aculeatus) ecotypes adapted to marine or freshwater environment. First, we identified genes with repeated expression or splicing divergence across ecotype pairs, and compared the genetic architecture and biological processes between parallelly expressed and parallelly spliced loci. Second, we analysed the extent to which parallel adaptation was reflected at gene expression and alternative splicing levels. Finally, we tested how the two axes of transcriptional variation differed in their potential for evolutionary change. Although both repeated differential splicing and differential expression across ecotype pairs showed tendency for parallel divergence, the degree of parallelism was lower for splicing than expression. Furthermore, parallel divergences in splicing and expression were likely to be associated with distinct cis-regulatory genetic variants and functionally unique set of genes. Finally, we found that parallelly spliced genes showed higher nucleotide diversity than parallelly expressed genes, indicating splicing is less susceptible to genetic variation erosion during parallel adaptation. Our results provide novel insight into the role of splicing in parallel adaptation, and underscore the contribution of splicing to the evolutionary potential of wild populations under environmental change.

Parallel evolution of integrated craniofacial traits in trophic specialist pupfishes.

Populations may adapt to similar environments via parallel or non-parallel genetic changes, but the frequency of these alternative mechanisms and underlying contributing factors are still poorly understood outside model systems. We used QTL mapping to investigate the genetic basis of highly divergent craniofacial traits between the scale-eater (Cyprinodon desquamator) and molluscivore (C. brontotheroides) pupfish adapting to two different hypersaline lake environments on San Salvador Island, Bahamas. We lab-reared F2 scale-eater x molluscivore intercrosses from two different lake populations, estimated linkage maps, scanned for significant QTL for 29 skeletal and craniofacial traits, female mate preference, and sex. We compared the location of QTL between lakes to quantify parallel and non-parallel genetic changes. We detected significant QTL for six craniofacial traits in at least one lake. However, nearly all shared QTL loci were associated with a different craniofacial trait within each lake. Therefore, our estimate of parallel evolution of craniofacial genetic architecture could range from one out of six identical trait QTL (low parallelism) to five out of six integrated trait QTL (high parallelism). We suggest that pleiotropy and trait integration can affect estimates of parallel evolution, particularly within rapid radiations. We also observed increased adaptive introgression in shared QTL regions, suggesting that gene flow contributed to parallel evolution. Overall, our results suggest that the same genomic regions may contribute to parallel adaptation across integrated suites of craniofacial traits, rather than specific traits, and highlight the need for a more expansive definition of parallel evolution.

Parallel Evolution at the Regulatory Base-Pair Level Contributes to Mammalian Interspecific Differences in Polygenic Traits.

Parallel evolution occurs when distinct lineages with similar ancestral states converge on a new phenotype. Parallel evolution has been well documented at the organ, gene pathway, and amino acid sequence level but in theory, it can also occur at individual nucleotides within noncoding regions. To examine the role of parallel evolution in shaping the biology of mammalian complex traits, we used data on single-nucleotide polymorphisms (SNPs) influencing human intraspecific variation to predict trait values in other species for 11 complex traits. We found that the alleles at SNP positions associated with human intraspecific height and red blood cell (RBC) count variation are associated with interspecific variation in the corresponding traits across mammals. These associations hold for deeper branches of mammalian evolution as well as between strains of collaborative cross mice. While variation in RBC count between primates uses both ancient and more recently evolved genomic regions, we found that only primate-specific elements were correlated with primate body size. We show that the SNP positions driving these signals are flanked by conserved sequences, maintain synteny with target genes, and overlap transcription factor binding sites. This work highlights the potential of conserved but tunable regulatory elements to be reused in parallel to facilitate evolutionary adaptation in mammals.

Genomics of hybrid parallel origin in Aquilegia ecalcarata.

BACKGROUND: The parallel evolution of similar traits or species provides strong evidence for the role of natural selection in evolution. Traits or species that evolved repeatedly can be driven by separate de novo mutations or interspecific gene flow. Although parallel evolution has been reported in many studies, documented cases of parallel evolution caused by gene flow are scarce by comparison. Aquilegia ecalcarata and A. kansuensis belong to the genus of Aquilegia, and are the closest related sister species. Mutiple origins of A. ecalcarata have been reported in previous studies, but whether they have been driven by separate de novo mutations or gene flow remains unclear. RESULTS: In this study, We conducted genomic analysis from 158 individuals of two repeatedly evolving pairs of A. ecalcarata and A. kansuensis. All samples were divided into two distinct clades with obvious geographical distribution based on phylogeny and population structure. Demographic modeling revealed that the origin of the A. ecalcarata in the Eastern of China was caused by gene flow, and the Eastern A. ecalcarata occurred following introgression from Western A. ecalcarata population. Analysis of Treemix and D-statistic also revealed that a strong signal of gene flow was detected from Western A. ecalcarata to Eastern A. ecalcarata. Genetic divergence and selective sweep analyses inferred parallel regions of genomic divergence and identified many candidate genes associated with ecologically adaptive divergence between species pair. Comparative analysis of parallel diverged regions and gene introgression confirms that gene flow contributed to the parallel evolution of A. ecalcarata. CONCLUSIONS: Our results further confirmed the multiple origins of A. ecalcarata and highlighted the roles of gene flow. These findings provide new evidence for parallel origin after hybridization as well as insights into the ecological adaptation mechanisms underlying the parallel origins of species.

Predictable and Divergent Change in the Multivariate P Matrix during Parallel Adaptation.

AbstractAdaptation to replicated environmental conditions can be remarkably predictable, suggesting that parallel evolution may be a common feature of adaptive radiation. An open question, however, is how phenotypic variation itself evolves during repeated adaptation. Here, we use a dataset of morphological measurements from 35 populations of threespine stickleback, consisting of 16 parapatric lake-stream pairs and three marine populations, to understand how phenotypic variation has evolved during transitions from marine to freshwater environments and during subsequent diversification across the lake-stream boundary. We find statistical support for divergent phenotypic covariance (P) across populations, with most diversification of P occurring among freshwater populations. Despite a close correspondence between within-population phenotypic variation and among-population divergence, we find that variation in P is unrelated to total variation in population means across the set of populations. For lake-stream pairs, we find that theoretical predictions for microevolutionary change can explain more than 30% of divergence in P matrices across the habitat boundary. Together, our results indicate that divergence in variance structure occurs primarily in dimensions of trait space with low phenotypic integration, correlated with disparate lake and stream environments. Our findings illustrate how conserved and divergent features of multivariate variation can underlie adaptive radiation.

Oncogenic composite mutations can be predicted by co-mutations and their chromosomal location.

Genetic heterogeneity in tumors can show a remarkable selectivity when two or more independent genetic events occur in the same gene. This phenomenon, called composite mutation, points toward a selective pressure, which frequently causes therapy resistance to mutation-specific drugs. Since composite mutations have been described to occur in sub-clonal populations, they are not always captured through biopsy sampling. Here, we provide a proof of concept to predict composite mutations to anticipate which patients might be at risk for sub-clonally driven therapy resistance. We found that composite mutations occur in 5% of cancer patients, mostly affecting the PIK3CA, EGFR, BRAF, and KRAS genes, which are common precision medicine targets. Furthermore, we found a strong and significant relationship between the frequencies of composite mutations with commonly co-occurring mutations in a non-composite context. We also found that co-mutations are significantly enriched on the same chromosome. These observations were independently confirmed using cell line data. Finally, we show the feasibility of predicting compositive mutations based on their co-mutations (AUC 0.62, 0.81, 0.82, and 0.91 for EGFR, PIK3CA, KRAS, and BRAF, respectively). This prediction model could help to stratify patients who are at risk of developing therapy resistance-causing mutations.

Comparative transcriptomics revealed parallel evolution and innovation of photosymbiosis molecular mechanisms in a marine bivalve.

Photosymbioses between heterotrophic hosts and autotrophic symbionts are evolutionarily prevalent and ecologically significant. However, the molecular mechanisms behind such symbioses remain less elucidated, which hinders our understanding of their origin and adaptive evolution. This study compared gene expression patterns in a photosymbiotic bivalve (Fragum sueziense) and a closely related non-symbiotic species (Trigoniocardia granifera) under different light conditions to detect potential molecular pathways involved in mollusc photosymbiosis. We discovered that the presence of algal symbionts greatly impacted host gene expression in symbiont-containing tissues. We found that the host immune functions were suppressed under normal light compared with those in the dark. In addition, we found that cilia in the symbiont-containing tissues play important roles in symbiont regulation or photoreception. Interestingly, many potential photosymbiosis genes could not be annotated or do not exhibit orthologues in T. granifera transcriptomes, indicating unique molecular functions in photosymbiotic bivalves. Overall, we found both novel and known molecular mechanisms involved in animal-algal photosymbiosis within bivalves. Given that many of the molecular pathways are shared among distantly related host lineages, such as molluscs and cnidarians, it indicates that parallel and/or convergent evolution is instrumental in shaping host-symbiont interactions and responses in these organisms.

Understanding evolutionary rescue and parallelism in response to environmental stress.

Evolutionary rescue, the process by which populations facing environmental stress avoid extinction through genetic adaptation, is a critical area of study in evolutionary biology. The order in which mutations arise and get established will be relevant to the population's rescue. This study investigates the degree of parallel evolution at the genotypic level between independent populations facing environmental stress and subject to different demographic regimes. Under density regulation, 2 regimes exist: In the first, the population can restore positive growth rates by adjusting its population size or through adaptive mutations, whereas in the second regime, the population is doomed to extinction unless a rescue mutation occurs. Analytical approximations for the likelihood of evolutionary rescue are obtained and contrasted with simulation results. We show that the initial level of maladaptation and the demographic regime significantly affect the level of parallelism. There is an evident transition between these 2 regimes. Whereas in the first regime, parallelism decreases with the level of maladaptation, it displays the opposite behavior in the rescue/extinction regime. These findings have important implications for understanding population persistence and the degree of parallelism in evolutionary responses as they integrate demographic effects and evolutionary processes.

Similar enzymatic functions in distinct bioluminescence systems: evolutionary recruitment of sulfotransferases in ostracod light organs.

Genes from ancient families are sometimes involved in the convergent evolutionary origins of similar traits, even across vast phylogenetic distances. Sulfotransferases are an ancient family of enzymes that transfer sulfate from a donor to a wide variety of substrates, including probable roles in some bioluminescence systems. Here, we demonstrate multiple sulfotransferases, highly expressed in light organs of the bioluminescent ostracod Vargula tsujii, transfer sulfate in vitro to the luciferin substrate, vargulin. We find luciferin sulfotransferases (LSTs) of ostracods are not orthologous to known LSTs of fireflies or sea pansies; animals with distinct and convergently evolved bioluminescence systems compared to ostracods. Therefore, distantly related sulfotransferases were independently recruited at least three times, leading to parallel evolution of luciferin metabolism in three highly diverged organisms. Reuse of homologous genes is surprising in these bioluminescence systems because the other components, including luciferins and luciferases, are completely distinct. Whether convergently evolved traits incorporate ancient genes with similar functions or instead use distinct, often newer, genes may be constrained by how many genetic solutions exist for a particular function. When fewer solutions exist, as in genetic sulfation of small molecules, evolution may be more constrained to use the same genes time and again.

Parallel evolution despite low genetic diversity in three-spined sticklebacks.

When populations repeatedly adapt to similar environments they can evolve similar phenotypes based on shared genetic mechanisms (parallel evolution). The likelihood of parallel evolution is affected by demographic history, as it depends on the standing genetic variation of the source population. The three-spined stickleback (Gasterosteus aculeatus) repeatedly colonized and adapted to brackish and freshwater. Most parallel evolution studies in G. aculeatus were conducted at high latitudes, where freshwater populations maintain connectivity to the source marine populations. Here, we analysed southern and northern European marine and freshwater populations to test two hypotheses. First, that southern European freshwater populations (which currently lack connection to marine populations) lost genetic diversity due to bottlenecks and inbreeding compared to their northern counterparts. Second, that the degree of genetic parallelism is higher among northern than southern European freshwater populations, as the latter have been subjected to strong drift due to isolation. The results show that southern populations exhibit lower genetic diversity but a higher degree of genetic parallelism than northern populations. Hence, they confirm the hypothesis that southern populations have lost genetic diversity, but this loss probably happened after they had already adapted to freshwater conditions, explaining the high degree of genetic parallelism in the south.

The prevalence of copy number increase at multiallelic copy number variants associated with cave colonization.

Copy number variation is a common contributor to phenotypic diversity, yet its involvement in ecological adaptation is not easily discerned. Instances of parallelly evolving populations of the same species in a similar environment marked by strong selective pressures present opportunities to study the role of copy number variants (CNVs) in adaptation. By identifying CNVs that repeatedly occur in multiple populations of the derived ecotype and are not (or are rarely) present in the populations of the ancestral ecotype, the association of such CNVs with adaptation to the novel environment can be inferred. We used this paradigm to identify CNVs associated with recurrent adaptation of the Mexican tetra (Astyanax mexicanus) to cave environment. Using a read-depth approach, we detected CNVs from previously re-sequenced genomes of 44 individuals belonging to two ancestral surfaces and three derived cave populations. We identified 102 genes and 292 genomic regions that repeatedly diverge in copy number between the two ecotypes and occupy 0.8% of the reference genome. Functional analysis revealed their association with processes previously recognized to be relevant for adaptation, such as vision, immunity, oxygen consumption, metabolism, and neural function and we propose that these variants have been selected for in the cave or surface waters. The majority of the ecotype-divergent CNVs are multiallelic and display copy number increases in cavefish compared to surface fish. Our findings suggest that multiallelic CNVs - including gene duplications - and divergence in copy number provide a fast route to produce novel phenotypes associated with adaptation to subterranean life.

The trichome pattern diversity of Cardamine shares genetic mechanisms with Arabidopsis but differs in environmental drivers.

Natural variation in trichome pattern (amount and distribution) is prominent among populations of many angiosperms. However, the degree of parallelism in the genetic mechanisms underlying this diversity and its environmental drivers in different species remain unclear. To address these questions, we analyzed the genomic and environmental bases of leaf trichome pattern diversity in Cardamine hirsuta, a relative of Arabidopsis (Arabidopsis thaliana). We characterized 123 wild accessions for their genomic diversity, leaf trichome patterns at different temperatures, and environmental adjustments. Nucleotide diversities and biogeographical distribution models identified two major genetic lineages with distinct demographic and adaptive histories. Additionally, C. hirsuta showed substantial variation in trichome pattern and plasticity to temperature. Trichome amount in C. hirsuta correlated positively with spring precipitation but negatively with temperature, which is opposite to climatic patterns in A. thaliana. Contrastingly, genetic analysis of C. hirsuta glabrous accessions indicated that, like for A. thaliana, glabrousness is caused by null mutations in ChGLABRA1 (ChGL1). Phenotypic genome-wide association studies (GWAS) further identified a ChGL1 haplogroup associated with low trichome density and ChGL1 expression. Therefore, a ChGL1 series of null and partial loss-of-function alleles accounts for the parallel evolution of leaf trichome pattern in C. hirsuta and A. thaliana. Finally, GWAS also detected other candidate genes (e.g. ChETC3, ChCLE17) that might affect trichome pattern. Accordingly, the evolution of this trait in C. hirsuta and A. thaliana shows partially conserved genetic mechanisms but is likely involved in adaptation to different environments.

Shrinking in the dark: Parallel endosymbiont genome erosions are associated with repeated host transitions to an underground life.

Microbial symbioses have had profound impacts on the evolution of animals. Conversely, changes in host biology may impact the evolutionary trajectory of symbionts themselves. Blattabacterium cuenoti is present in almost all cockroach species and enables hosts to subsist on a nutrient-poor diet. To investigate if host biology has impacted Blattabacterium at the genomic level, we sequenced and analyzed 25 genomes from Australian soil-burrowing cockroaches (Blaberidae: Panesthiinae), which have undergone at least seven separate subterranean, subsocial transitions from above-ground, wood-feeding ancestors. We find at least three independent instances of genome erosion have occurred in Blattabacterium strains exclusive to Australian soil-burrowing cockroaches. These shrinkages have involved the repeated inactivation of genes involved in amino acid biosynthesis and nitrogen recycling, the core role of Blattabacterium in the host-symbiont relationship. The most drastic of these erosions have occurred in hosts thought to have transitioned underground the earliest relative to other lineages, further suggestive of a link between gene loss in Blattabacterium and the burrowing behavior of hosts. As Blattabacterium is unable to fulfill its core function in certain host lineages, these findings suggest soil-burrowing cockroaches must acquire these nutrients from novel sources. Our study represents one of the first cases, to our knowledge, of parallel host adaptations leading to concomitant parallelism in their mutualistic symbionts, further underscoring the intimate relationship between these two partners.

Pervasive mimicry in flight behavior among aposematic butterflies.

Flight was a key innovation in the adaptive radiation of insects. However, it is a complex trait influenced by a large number of interacting biotic and abiotic factors, making it difficult to unravel the evolutionary drivers. We investigate flight patterns in neotropical heliconiine butterflies, well known for mimicry of their aposematic wing color patterns. We quantify the flight patterns (wing beat frequency and wing angles) of 351 individuals representing 29 heliconiine and 9 ithomiine species belonging to ten color pattern mimicry groupings. For wing beat frequency and up wing angles, we show that heliconiine species group by color pattern mimicry affiliation. Convergence of down wing angles to mimicry groupings is less pronounced, indicating that distinct components of flight are under different selection pressures and constraints. The flight characteristics of the Tiger mimicry group are particularly divergent due to convergence with distantly related ithomiine species. Predator-driven selection for mimicry also explained variation in flight among subspecies, indicating that this convergence can occur over relatively short evolutionary timescales. Our results suggest that the flight convergence is driven by aposematic signaling rather than shared habitat between comimics. We demonstrate that behavioral mimicry can occur between lineages that have separated over evolutionary timescales ranging from <0.5 to 70 My.

Parallel evolution in mosquito vectors - a duplicated esterase locus is associated with resistance to pirimiphos-methyl in An. gambiae.

The primary control methods for the African malaria mosquito, Anopheles gambiae, are based on insecticidal interventions. Emerging resistance to these compounds is therefore of major concern to malaria control programmes. The organophosphate, pirimiphos-methyl, is a relatively new chemical in the vector control armoury but is now widely used in indoor residual spray campaigns. Whilst generally effective, phenotypic resistance has developed in some areas in malaria vectors. Here, we used a population genomic approach to identify novel mechanisms of resistance to pirimiphos-methyl in Anopheles gambiae s.l mosquitoes. In multiple populations, we found large and repeated signals of selection at a locus containing a cluster of detoxification enzymes, some of whose orthologs are known to confer resistance to organophosphates in Culex pipiens. Close examination revealed a pair of alpha-esterases, Coeae1f and Coeae2f, and a complex and diverse pattern of haplotypes under selection in An. gambiae, An. coluzzii and An. arabiensis. As in Cx. pipiens, copy number variation seems to play a role in the evolution of insecticide resistance at this locus. We used diplotype clustering to examine whether these signals arise from parallel evolution or adaptive introgression. Using whole-genome sequenced phenotyped samples, we found that in West Africa, a copy number variant in Anopheles gambiae is associated with resistance to pirimiphos-methyl. Overall, we demonstrate a striking example of contemporary parallel evolution which has important implications for malaria control programmes.