Evolutionary Applications

Biological control agents often experience demographic bottlenecks during introduction, which can reshape genetic diversity and inbreeding pattern influencing establishment and long-term ecological success in the introduced populations. The leaf-feeding beetle Calligrapha (Zygogramma) bicolorata, introduced in multiple countries across the globe to control the weed Parthenium hysterophorus, provides an opportunity to examine how introduction bottleneck and post-introduction breeding practices shape genomic variation and inbreeding patterns. We analyzed whole-genome variation in the introduced beetle population in India by sampling six regions encompassing its current distribution in the country. Using genome-wide variation data, we assessed population structure, genetic diversity, and inbreeding patterns across regions, and inferred historical changes in effective population size to reconstruct post-introduction demographic trajectories. The analyses reveal subtle genetic structure across regions, with overall genetic diversity relatively low compared to other invasive and biocontrol insects. Inbreeding patterns vary among populations, with some regions exhibiting higher cumulative runs of homozygosity than others. Notably, regions subjected to intensive propagation of beetle populations show elevated signature of inbreeding alongside reduced historical effective population size. These results underscore the dynamic genomic consequences of biocontrol introduction and subsequent breeding practices, providing insight into the evolutionary trajectories of introduced biocontrol agents.

1Effective population size (Ne) is a critical parameter for evaluating the evolutionary and persistence potential of endangered populations and for designing sustainable conservation strategies. Captive breeding and release programs are widely used across taxa to reduce risk of extinction when natural reproduction is insufficient or no longer possible, making it essential to assess their consequences. We used the case study of the landlocked Saimaa salmon (Salmo salar), one of the most critically en-dangered salmonid populations in Europe, with unique evolutionary significance due to its isolation from other populations since the last glaciation. Using long-term demographic data (1969-2024) from wild-caught founders of a captive breeding and release program, we estimated the effective population size under multiple scenarios of variance in reproductive success. Across scenarios, Ne ranged from 33 to 81 individuals, representing 32%-75% of the census size. Captive breeding practices aimed at equalizing parental contributions during fertilization and early life stages increased Ne by 12% compared to natural reproductive conditions. However, variation in survival after early developmental stages, typically beyond direct management control, remained a key determinant of Ne. Despite recent increases in the number of founders, the population remains genetically vulnerable due to historical bottlenecks. These results highlight that while captive breeding programs can partially mitigate genetic risks, their effectiveness depends critically on both controlled and uncontrolled sources of variance in reproductive success. Strengthening such programs may require combining breeding management with habitat restoration and, where appropriate, genetic rescue to ensure the long-term evolutionary potential of such unique and endangered populations.

The European flat oyster (Ostrea edulis), like numerous other oyster species, has been exploited for millennia and cultivated and translocated for centuries. Following a severe population decline, and in the context of ongoing conservation and restoration programs, genetic considerations must now be addressed to avoid mistakes. The objective of our study was to complement population genetic studies conducted at various scales along European coasts. Our sampling primarily targeted the French Atlantic, English Channel, and Mediterranean coasts, aiming to provide a fine-scale genetic characterization of populations in these regions. By integrating SNP array and low-coverage sequencing datasets, we obtained a comprehensive overview of the population genetic structure of Ostrea edulis across western Europe. Most previously identified clusters in Western Europe were confirmed. In France, populations assigned to these clusters exhibited notable within-patch homogeneity. However, two key findings emerged: (1) an extensive overlap zone between the Atlantic and western Mediterranean clusters, spanning at least from southern Portugal to southern France, and (2) the detection of a novel, clearly distinct cryptic cluster east of the English Channel, whose geographic range remains to be better delineated. These insights are critical for informing management decisions, particularly as restoration and conservation plans are currently being implemented across the species range.

Long-term persistence of managed species will depend, in part, on whether the species harbors the physiological or genetic potential to adjust to warming temperatures, and whether relevant genetic variation is modified by management practices. The critically endangered Delta Smelt (Hypomesus transpacificus) is intensively managed, but little is known about the presence of genetic variation for resistance to elevated temperature, which will be important to maintain for their persistence in a rapidly warming future. Using a pedigree and whole genome sequencing data, we characterized the genetic variation and genomic architecture for CTMax (as a metric of upper thermal tolerance) across control and elevated rearing temperatures, alongside covarying traits (body size, degree of hatchery ancestry). Warmer rearing temperatures increased CTMax through acclimation but also resulted in reduced additive genetic variation for the trait, which could constrain adaptation under thermal stress. We found that larger fish had reduced CTMax, although this effect was diminished at elevated temperatures. We observed modest heritability for CTMax at rearing temperatures of 15{degrees}C and 18{degrees}C (0.26 and 0.16, respectively), but only a limited number of loci were identified that had consistent effects on CTMax across rearing temperatures. Instead, the genomic basis of thermal tolerance was highly dependent on rearing temperature (many loci detected with a GxE effect). The influence of domestication selection was indicated by changes in allele frequency, and divergence in upper thermal tolerance and plasticity, between low and high hatchery ancestry groups. Minimal overlap between loci associated with domestication and CTMax suggests that these traits possess separate genetic underpinnings. Knowledge of genetic variation supporting ecologically relevant physiological variation may be useful for refuge management and may inform supplementation in an ever-warming environment.

Japanese chum salmon (Oncorhynchus keta) populations supported by one of the worlds largest hatchery programs have experienced severe declines in marine survival and abundance. To evaluate long-term changes in reproductive traits, we analyzed temporal and geographic variation in female fork length (FL) and egg size from 13 hatchery-enhanced rivers in Japan during 1999-2019. Females from northern rivers tended to have smaller FL but produced larger eggs, indicating persistent latitudinal differentiation in reproductive traits. Despite this geographic structure, both FL and egg size declined synchronously among rivers over time. Using nationwide annual means, exponential decay analyses showed that egg size declined by approximately 2% per generation, whereas marine survival and population abundance declined by approximately 22% per generation. Mean egg size was positively correlated with subsequent marine survival, explaining 45% of the variation in return rates. Mean female FL explained 64% of the variation in mean egg size. Exponential decay rates estimated from standardized variables were nearly identical among egg size, marine survival, and chum salmon abundance, suggesting common underlying drivers of long-term decline.

Genetic interventions to modify wild population densities are typically framed around pest suppression, with parallel approaches for boosting beneficial or endangered populations remaining largely undeveloped. Imposing a sustained but non-eliminative genetic load could in principle address both objectives, but existing designs rely on genes with intermediate fitness effects whose loads are difficult to predict under field conditions. Here we describe engineered balanced lethal systems, in which CRISPR-based gene drive establishes two complementing recessive-lethal alleles at a single locus, producing a sustained 50% load through Mendelian segregation. Modelling shows these systems spread from small releases, and that the resulting population-level consequences depend on density regulation and on the timing of lethality: the same 50% load can suppress pests, boost populations of beneficial or endangered species, dampen boom-bust cycles, or raise effective population size. Additional systems at independent loci scale the effect in stepwise increments, and a split-drive variant localises it geographically. These results demonstrate that gene drives imposing genetic load can be expanded beyond elimination, to support and preserve beneficial and endangered populations.

Genomic forecasting approaches based on genotype-environment associations (GEAs) are increasingly used to estimate genomic offsets (GOs), which predict population maladaptation and extinction risk under current or future climatic conditions. Despite their widespread use, only a subset of studies have evaluated how accurately GOs predict (mal)adaptation, limiting their interpretation and application in policy and management. Here, we used GEA analyses to estimate GOs for past, present, and future climates in Lycaeides butterflies, focusing on the causes of variation in GOs among populations and their relationships with demographic parameters inferred from population genomic data. Using multivariate linear regression and genotyping-by-sequencing data from 42 Lycaeides populations (922 butterflies), we found that mean annual temperature, cumulative annual precipitation, and hybridization history together explained 47.6% of variation in genome-wide allele frequencies. Genomic offsets differed substantially among populations and across past, present, and future climates, with evidence for increasing maladaptation under more distant future climate scenarios. We found no relationship between GOs for present climates and contemporary effective population size. In contrast, genetic diversity, which reflects long-term effective population size, and local rates of gene flow together explained 27.3% of variation in contemporary GOs. Populations with higher genetic diversity and more gene flow exhibited lower GOs, consistent with the hypothesis that genetic diversity enhances adaptive capacity and that gene flow may introduce adaptive alleles. Overall, our results support the utility of GO predictions, particularly when validated with independent measures of adaptation, while cautioning against simplistic interpretations of GO as a direct measure of maladaptation in conservation and management contexts.

Genomic offset (GO) is increasingly used to predict population maladaptation risk under climate change, with larger offsets assumed to indicate greater vulnerability. Despite rapid adoption in conservation planning, it remains unclear how sensitive GO estimates are to key methodological choices, including SNP set composition, genotype-environment association (GEA) methods, and the specific GO metric used. Empirical validation against observed population dynamics also remains limited. Here, we evaluate the methodological robustness and predictive performance of GO using multidecadal demographic monitoring data from Lycaeides butterflies, a system with short generation times and high fecundity that may facilitate rapid adaptive responses. GO estimates were broadly consistent across SNP sets, regardless of composition or size, with climate-associated and randomly selected SNPs yielding largely concordant values. Consistency across GEA methods was moderate and depended on the SNP set used. In contrast, GO metrics differed substantially in the magnitude of maladaptation estimated, suggesting they capture distinct biological signals and should not be treated as interchangeable. Crucially, GO was a poor predictor of observed population trends, regardless of SNP set composition, GO metric, or GEA method, both at sites used to fit GEA models and when extrapolated to independent demographic sites. These findings suggest that, while GO provides a valuable conceptual framework for assessing potential maladaptation, its quantitative estimates and predictive power are sensitive to methodological choices and species-specific biological context. We therefore urge careful alignment of GO metric assumptions with conservation objectives, along with rigorous empirical validation, before GO estimates are used to inform management decisions.

Habitat fragmentation creates small, isolated populations vulnerable to inbreeding, genetic drift, and high genetic load. For conservation management, it is essential to distinguish contemporary landscape resistance from historical demographic processes as drivers of these genetic patterns, especially for conservation priority regions such as northeast India which intersects two major tiger conservation landscapes. We studied the genetic structure and landscape connectivity of tigers across four protected areas in northeast India: Kaziranga, Manas, Orang, and Nameri, using faecal samples. From 741 samples collected over two field seasons, we identified 654 confirmed tiger specimens. Using methylation-based enrichment and ddRAD-seq of 176 samples, we generated a high-quality dataset of 3091 SNPs across 44 individuals. Population structure analyses identified three genetically distinct clusters: Kaziranga-Nameri, Manas, and Orang. Isolation-by-distance and landscape resistance explained 13% and 17% of the observed genetic divergence, respectively, with human settlements influencing gene flow. Orangs pronounced divergence from Kaziranga, despite geographic proximity and corridors, suggests a post-bottleneck founder effect, as evidenced by reduced heterozygosity (Ho = 0.24), nucleotide diversity (pi = 0.24), and effective population size (Ne = 1.3). These findings reveal that demographic and genetic recovery can decouple: Orangs population has recently grown, yet genome-wide evidence shows ongoing genetic erosion that monitoring has not detected. Similar patterns have been reported in other Indian tiger populations, indicating that such decoupling may be systemic. Target 4 of the Kunming-Montreal Global Biodiversity Framework requires explicit genetic diversity monitoring; this study demonstrates that non-invasive genomics can operationalise that mandate at a conservation-relevant scale.

Thermal history provenancing can guide the choice of parental broodstock for selective breeding of corals from distinct reefs and has been proposed as an intervention for enhancing climate resilience. However, the genetic and molecular mechanisms underlying resultant offspring responses to heat stress, particularly during early life stages, remain poorly understood. Here, we generated Acropora tersa larvae and recruits by crossing parental colonies from the historically warmer Martin Reef and cooler Davies Reef and assessed the effects of within- and between-reef crosses on genetic diversity and transcriptional responses to heat stress. Genome-wide single nucleotide polymorphism analyses showed that broodstock from Martin and Davies Reefs were weakly differentiated (FST = 0.008) and exhibited comparable heterozygosity, as did all larval offspring groups. Transcriptomic analyses of recruits exposed to heat stress (32 {degrees}C for 36 days) revealed that both within- and between-reef offspring groups activated conserved stress-response pathways, with seven genotype-independent heat-responsive genes detected across all offspring groups. Differential expression and enrichment analyses showed induction of defence, protein homeostasis, intracellular transport, and metabolic processes alongside repression of growth- and signalling-related functions, consistent with the Type A General Coral Stress Response. Taken together, these findings suggest that the benefits of thermal history provenancing-informed selective breeding may be limited in low-differentiation systems and that targeted pre-screening of broodstock may help capture functional genetic variation relevant to restoration applications.

O_LIEffective conservation of highly migratory species requires understanding genetic structure across breeding populations and access high{square}resolution markers capable of assigning individuals from mixed aggregates (e.g. bycatch or new nesting sites) to their natal origins. Genomic approaches provide unprecedented resolution but add methodological challenges; thus, it is essential to first build a genomic baseline from known breeding areas and then evaluate strategies for assigning unknown individuals. C_LIO_LITo address this, we used 2b-RAD sequencing, a genomic reduction technique useful for degraded DNA, and loggerhead turtles as a case study. This species shows philopatric breeding, while juveniles and adults form mixed aggregations in foraging grounds. C_LIO_LIOur results highlight the importance of building baselines that include all potential source populations contributing to mixed aggregations. We detected hierarchical genetic differentiation and high resolution and successfully assigned the natal origin of 124 unknown individuals from four Mediterranean foraging grounds. These grounds showed distinct source contributions, and comparisons with previous studies suggest possible temporal shifts in stock composition. C_LIO_LIWe provide a comprehensive genomic baseline for individual assignment of Altanto-Mediterranean loggerhead turtles of unknown natal origin and a general framework for identifying population-specific threats in highly migratory species. C_LI

Understanding population structure is critical for effective fisheries management in species with complex life histories and variable recruitment. Southern flounder (Paralichthys lethostigma) is a valuable flatfish species with declining populations in the Southeast United States. Improved management may depend on a better understanding of fine-scale and temporal population genetic structure in this region; however, such structure remains poorly characterized. To address our lack of understanding of the spatial and temporal population structure of this important species, we used double digest reduced-representation genome sequencing (ddRADSeq) on juveniles from estuaries in North Carolina and Texas between 2014 and 2023. We found significant genetic differentiation between the Gulf of Mexico and Atlantic populations, supporting the management of these regions as distinct stocks. By contrast, we detected significant variance in genetic structure within Texas and North Carolina populations that was not consistent across sampling years between estuaries in close proximity. The population genetic structure of southern flounder suggests significant, temporally variable genetic differences within estuarine locations that may result from variation in larval dispersal and recruitment patterns. Our findings highlight the value of integrating fine-scale, multi-year genetic data to capture temporal dynamics and avoid misleading conclusions based on single-year or broad-scale sampling.

Secondary endosymbioses typically involve photosynthetic gain-of-function, at least initially. For Neoparamoeba perurans, the agent of Amoebic Gill Disease in Atlantic salmon, the evolutionary significance of its non-photosynthetic kinetoplastid endosymbiont, Perkinsela, remains a mystery. While such endosymbionts usually mirror organellar uniparental inheritance, departures from strict vertical transmission can significantly impact holobiont evolution and the spread of traits like drug resistance. However, genomic analysis of N. perurans is hampered by continual traffic of bacteria in and out of the amoeba, limiting our understanding of its population dynamics. Here, we developed a dual-target AmpSeq panel from a draft N. perurans genome, enabling simultaneous genotyping of host and symbiont directly from xenic biological samples. Analysis of 58 North Atlantic isolates revealed a striking asymmetry in population structure: the amoeba host forms a diverse, panmictic population with low linkage disequilibrium, whereas Perkinsela exhibits lower diversity and strong clonality. While cophylogenetic analyses confirm a global signal of vertical fidelity, fine-scale genotyping reveals frequent shuffling of host-symbiont pairs. These results suggest that strict host-symbiont co-inheritance is not absolute, perhaps reflecting a system characterised by long-term vertical stability punctuated by intermittent reassortment. Ultimately, we validate AmpSeq as a robust, scalable tool for decoupling complex host-symbiont trajectories and monitoring multi-partner disease dynamics in aquaculture environments.

The deployment of clothianidin-based insecticide formulations in malaria vector control has highlighted the capacity of Anopheles funestus to displace more susceptible mosquito species in treated areas and to rapidly evolve resistance under selection pressure. Metabolic detoxification, together with structural and genetic changes in nicotinic acetylcholine receptors (nAChRs), the primary molecular targets of neonicotinoids, can reduce insecticide efficacy. Here, we characterized amino acid substitutions across all 11 nAChR subunits in An. funestus to assess standing variation that may facilitate adaptive responses to chemical exposure. Using whole-genome sequencing data from 656 mosquitoes sampled in 13 African countries, we found marked contrasts in the distribution of nonsynonymous variants among nAChR subunits. Most subunits are strongly constrained and carry no missense variants, whereas two loci (3 and 7) display three geographically widespread amino acid substitutions across the continent. In contrast, 9 and {beta}2 accumulate dozens of nonsynonymous mutations occurring at intermediate to high frequencies, including within domains involved in orthosteric ligand binding and channel gating. Genetic differentiation at nAChR loci among populations from different countries is low to moderate, although several nonsynonymous mutations display high FST values consistent with geographic structuring. These results highlight relaxed constraint on two subunits that may provide opportunities for evolutionary diversification within a conserved family of multimeric receptor assemblies. Such diversification has not been observed in vector species displaced by An. funestus in indoor residual spraying areas, and the potential implications for reduced sensitivity to neonicotinoids are discussed.

Despite being two of the most commercially important flatfish (order Pleuronectiformes) in Europe, little is known of the population structure of common sole Solea solea and European plaice Pleuronectes platessa. To address this gap, we generated full-genome resequencing data for 244 sole and 189 plaice in the Celtic Sea and western English Channel region to analyse both neutral and adaptive loci and quantify population processes, such as reproductive isolation or adaptive differentiation in each species. For sole, there was no evidence of reproductive isolation or population structure at neutral loci. There was, however, adaptive differentiation as adaptive loci indicated two subpopulations, with separation in the western English Channel. This is consistent with previous studies using RAD-seq and gene-linked SNPs. For plaice, there was no evidence of population structure at either neutral or adaptive loci in the Celtic Seas and Western English Channel region. However, when considering a larger geographical area and utilising previously published genomic data, three distinct populations of plaice were identified (Iceland; North Sea, Kattegat and Western Baltic; Celtic Sea and western English Channel), with clear reproductive isolation indicated by neutral loci and adaptive differentiation indicated by adaptive loci. Moreover, three large chromosomal inversions were identified, which differed in their frequency between regions. These large structural variants represent putative key regions for adaptive differentiation. This study shows the benefit from quantifying neutral and adaptive loci to better understand population structure and genetic diversity of commercially important fish.

Population-specific immunity can drive variation in infection outcomes, but studying immune variation in the wild is challenging because exposure histories are unknown. Comparing wild populations with those reared in a common environment can disentangle genetic and environmental drivers of immunity. We applied this approach in freshwater threespine stickleback, where populations vary in their use of intraperitoneal fibrosis to defend against the helminth parasite Schistocephalus solidus. We combined a 46-lake immune survey with a common garden experiment using 20 representative populations to examine variation in fibrosis and infection. Laboratory assays included exposures to live tapeworms and immune challenges with tapeworm proteins and aluminum phosphate (Alum). We found heritable variation in both constitutive fibrosis and inducible fibrosis. Inducible responses to tapeworms were associated with lake environmental conditions, with fish from more eutrophic-like lakes showing stronger fibrosis induction than those from more oligotrophic-like lakes. Together, these results show how integrating wild immune variation with common garden experiments can reveal novel heritable defenses and link their evolution to ecological variation.

Population connectivity and adaptive gene flow in disease vectors can shape the emergence and spread of insecticide resistance, with direct implications for control strategies such as insecticide spraying or the use of bed nets for malaria control. Using whole-genome sequencing, we first resolved the geographic population structure of the major but understudied malaria vector, Anopheles arabiensis, across the East African region, including the geographically diverse country of Ethiopia. We then assessed evidence for adaptive gene flow of insecticide resistance alleles across the region. Within Ethiopia, Central Rift Valley populations flanked by mountainous terrain were subject to restricted gene flow, although higher connectivity with the southwestern populations suggests an intermediate point of genetic exchange with the rest of Ethiopia. Anopheles arabiensis from western and northernmost Ethiopia were connected to populations from a similarly arid environment in Turkana - Kenya. Furthermore, broad-scale analysis revealed that populations from the rest of Kenya were connected with those from Uganda and Tanzania, but finer-scale analysis revealed more subtle structuring along the Rift Valley flanks, underscoring the role of landscape features in shaping patterns of gene flow. Adaptive gene flow analyses of diplotype clustering revealed that resistance alleles such as Cyp6aa/p and Gste2 copy number variants (CNV) were widely spread across East Africa despite the geographical population structuring we observed. At the Gste2 locus, An. arabiensis from Ethiopia and Kenya carried a newly annotated CNV spanning chromosome position 3R:28,596,832-28,606,222 linked to the non-synonymous SNP V47L, which was only at a low frequency in Kenya. Collectively, our findings demonstrate that An. arabiensis is subject to transboundary movement of resistance alleles, highlighting the need for coordinated cross-country vector management. Although areas of high connectivity may challenge genetic control technologies such as gene drives, more isolated populations may provide opportunities for targeted deployment. SignificanceMosquitoes movement across landscapes determines how insecticide resistance spreads, yet the genomic connectivity of Anopheles arabiensis populations in East Africa is understudied. Using whole genome data, we show that geographic features such as the Rift Valley restrict gene flow in Ethiopia, with western and northernmost Ethiopia connecting to Turkana, while Kenyan populations outside Turkana connect further south with Tanzania and Uganda. Despite these geographic barriers, resistance variants at genes such as Cyp6aa/p and Gste2 spread widely through adaptive gene flow, including a newly annotated CNV allele at Gste2 in Ethiopia and Kenya. These findings demonstrate that resistance alleles move across national borders, underscoring the need for coordinated regional vector management. They also highlight that while broader connectivity may limit genetic control tools such as gene drive, more isolated populations offer opportunities for targeted deployment.

Whether global warming drives thermal adaptation in arboviral vectors is an open question with direct consequences for forecasting transmission risk and informing effective vector control strategies. We subjected Aedes albopictus to experimental evolution (EE) under a tropical thermal regime (32{degrees}C/26{degrees}C) for three years and tracked fitness and behavioral traits, energy reserves and the fat body transcriptional profile across replicate lines at defined generational intervals. We show that mosquitoes strongly acclimate, trading longevity for accelerated development and increased reproduction. Over 15-20 generations of EE, mosquitoes gradually adjusted adult longevity to values of controls, reproductive traits reduced while maintaining values higher than those of controls, and the transcriptome converged to a distinct metabolic state. Upon relaxing thermal selection, half of the transcriptional changes of warm-evolved mosquitoes, along with traits of juvenile development and adult lifespan, reversed to values of controls, indicating a plastic basis. In contrast, progeny per female and egg freeing point maintained warm-evolved values upon relaxing thermal selection. Moreover, the mean and variance of 250 differentially expressed genes showed a significant correlation in warm-evolved mosquitoes, with a considerable reduction of expression variance upon relaxing thermal selection, signatures consistent with selection acting on a polygenic trait architecture. Modelling of the net reproductive rate across generations showed that egg-to-adult viability, not fecundity or longevity, is the primary driver of reproductive success under warm evolution. Our findings have immediate methodological and ecological implications: clinal studies or single-generation exposures risk attributing to adaptation changes driven by phenotypic plasticity, acclimation, genetic drift or population structure.

RDL (Resistance to dieldrin) is a GABA-gated chloride channel that was first described as target of the insecticide dieldrin. Despite dieldrin being discontinued for decades because of its environmental per-sistence and health concerns, Rdl resistance mutations (A296S, A296G) continue at high frequencies in natural populations of the malaria mosquito Anopheles gambiae complex across Africa, suggesting a selective advantage. We have recently shown that RDL acts as a critical modulator of mosquito auditory sensitivity. Because acoustic recognition is essential for mate acquisition in An. gambiae, we hypothesized that these mutations confer a pleiotropic effect on mating success in the field, mediated through altered acoustic sensitivity, with potential consequences for sexual selection. We first provide laboratory evidence that resistance mutations enhance auditory behaviours of An. gambiae and show that the effect of environmental noise on mating success depends on the male Rdl genotype. We then conduct field collections in the city of Bangui (Central African Republic) and surrounding rural areas, revealing the presence of Rdl resistant alleles and their association with the urban environment, and within the city, with the noisiest locations. We also show decreased mating success of susceptible females with increasing noise levels, suggesting detrimental effects. Together, our findings support that Rdl resistance mutations enhance auditory function and mating success in acoustically challenging environments. We propose that this auditory advantage may contribute, together with other selective pressures such as cross-selection by other insecticides, to the persistence of these alleles in nature and may facilitate urban colonization by malaria vectors. Our study reveals, for the first time, an unintended evolutionary consequence of insecticide use, where a resistance mutation has been co-opted to enhance sensory performance and ecological adaptation, with significant implications for vector management strategies.

Morus rubra is native to the eastern United States, with its range extending into the Upper Midwest and southern Ontario, Canada. Its present distribution suggests that past glacial events in North America may have influenced the genetic structure of populations at the species northwestern range boundary. This study assessed genetic variation among six M. rubra populations believed to have experienced postglacial colonization using published nuclear microsatellite markers and sequences from the chloroplast trnL-trnF region. Five nuclear microsatellite markers previously developed for M. alba were successfully transferred to M. rubra, while the chloroplast trnL-trnF region provided an additional marker for evaluating chlorotype diversity. Nuclear microsatellite diversity was higher in southern unglaciated populations than in northern glaciated populations, a pattern consistent with the observed distribution of chlorotype diversity. Together, these results support ancient founder effects associated with leading-edge expansion following glacial recession and suggest that postglacial colonization contributed to the present-day genetic structure of M. rubra at its northwestern range boundary. Because M. rubra hybridizes with the naturalized invasive M. alba, reduced genetic diversity in marginal populations may increase their vulnerability to genetic swamping. The markers characterized in this study provide useful tools for population genetic research in Morus, and the findings have important implications for the conservation and management of marginal and threatened M. rubra populations in the Upper Midwest.