Evolutionary Applications

Hybridization between invasive and native species can generate novel genetic variation with ecological and evolutionary consequences, yet introgression often remains cryptic. We investigated natural hybridization between the invasive pinewood nematode Bursaphelenchus xylophilus, the causal agent of pine wilt disease, and native Bursaphelenchus mucronatus in Japan. Using multiplexed inter-simple sequence repeat genotyping by sequencing, we genotyped 342 field-collected nematodes and then conducted pedigree-calibrated analysis using HIest v2.0, based on 48 highly informative diagnostic single-nucleotide polymorphisms (SNPs), followed by ADMIXTURE, complementary NeighborNet and principal component analyses of a broader 2,756-SNP dataset. The results identified nine high-confidence hybrids (2.6%) across two consecutive years (2023-2024), including relatively balanced F1-like individuals and strongly parentally biased advanced backcross-like individuals, demonstrating recurrent, multigenerational introgression. Four hybrids (44.4%) were undetectable using a single-locus diagnostic marker, showing that advanced introgression can escape conventional surveillance. Chemotaxis assays detected no volatile-mediated pre-mating discrimination, whereas post-mating barriers were incomplete and asymmetric. Crosses with B. xylophilus as the maternal parent showed reduced hatchability, particularly with B. mucronatus mucronatus, while reciprocal crosses generally retained higher hatchability, although some showed reduced fecundity. These results provide a mechanistic basis for natural gene flow in a recent secondary contact zone. Because B. mucronatus kolymensis is widespread in Europe and produces viable hybrid offspring with B. xylophilus in both directions, pathogen establishment could create new opportunities for introgression. Our findings reveal cryptic, multigenerational introgression between an invasive forest pathogen and its native congener, highlighting the importance of genomic surveillance for forest disease management and invasion-risk assessment under climate change.

Many economically important insect pests reproduce through asexual or partially asexual life cycles, yet how reproductive mode influences insecticide resistance management remains unclear. The choice of resistance management strategy has been suggested to differ for sexual and asexual pests. For instance, current IRAC guidance suggests that pesticide mixtures are less effective in non-mating pests than in sexually reproducing populations. Here, stochastic evolutionary simulations are used to compare resistance evolution under sequences and mixtures across four reproductive modes observed in pests of economic importance: sexual reproduction, obligate parthenogenesis, cyclical parthenogenesis and haplodiploidy. Contrary to current expectations, mixtures are not disadvantaged in asexual populations and, in some cases, lead to delayed resistance evolution compared to sexually reproducing populations. These differences arise as the result of reduced genetic recombination which constrained the assembly and spread of multi-resistant genotypes. Overall, these findings suggest that mixtures remain a viable resistance management strategy for pests with asexual reproduction.

Habitat fragmentation and destruction in old-growth forests represents a primary threat to specialist species, frequently resulting in the erosion of genetic diversity. The Hermit Beetle, Osmoderma barnabita, is a protected relic of primeval forests and an obligate saproxylic specialist whose larval development depends exclusively on the wood-mould of hollow trees. This study provides the first high-resolution, genome-wide assessment of O. barnabita across seven Polish forest complexes using double-digestion Restriction-site Associated DNA sequencing data from 49 individuals. Following rigorous quality control and reference bias assessment, we evaluate contemporary population structure. To distinguish between evolutionary processes, we isolated 2,920 putatively adaptive outliers from the neutral background, evaluating both neutral and adaptive genomic signals. The analyses revealed four distinct genetic clusters, confirming that the Polish metapopulation is severely subdivided. Crucially, the westernmost population in the Lower Silesian Forest emerged as the most divergent evolutionary unit (FST up to 0.360), suggesting potential hybrid origin or long-term isolation. We observed a striking linear correlation (R2=0.986) between neutral diversity and adaptive potential, indicating that genetic drift in small and isolated forest fragments is actively depleting the variation required for long-term persistence of this species. Our findings demonstrate significant gene flow restriction across all sites, suggesting that current conservation frameworks fail to encompass these highly differentiated lineages. To mitigate this genetic vulnerability, we recommend reclassifying these clusters as independent Management Units and Evolutionary Significant Units. Maintaining forest continuity and protecting local micro-habitats are essential to prevent further isolation and preserve the genomic integrity of this saproxylic flagship species.

The box tree moth (Cydalima perspectalis) is an invasive pest of boxwood plants (Buxus spp.) that has rapidly expanded across Europe and, since its first detection in Canada in 2018, across North America. Understanding its introduction pathways and early population structure is critical for effective surveillance and management. We integrated genome-wide single nucleotide polymorphism (SNP), mitochondrial cytochrome c oxidase I (COI), and morphological data to investigate the early invasion history of C. perspectalis across North America. Genome-wide analyses revealed pronounced genetic differentiation between North American and European populations, together with substantial genetic structure within North America. Delaware was genetically distinct from other North American populations and showed greater affinity with European populations, consistent with a separate introduction history. In contrast, New York, Massachusetts, Michigan, Ohio, and Ontario showed greater genetic similarity, suggesting shared ancestry and/or regional connectivity, whereas Virginia and West Virginia exhibited additional differentiation. Mitochondrial data supported shared ancestry among most North American populations, while Delaware shared a haplotype group with the Croatian European population. D-statistic analyses identified asymmetric allele sharing among several North American and European populations, consistent with multiple introductions and subsequent admixture. Morphometric analyses revealed significant geographic variation in several body and wing traits. Together, these complementary datasets indicate that multiple introductions, regional genetic structuring, and admixture have shaped the North American invasion. These findings highlight the value of integrating genomic approaches with nursery and border surveillance to identify introduction pathways and inform targeted containment and long-term management of this emerging horticultural pest.

Dermo disease caused by the protist Perkinsus marinus poses a major threat to Eastern oyster aquaculture. We previously conducted genomic selection for dermo resistance and found increased effectiveness over phenotypic selection. Here, we report improved genomic predictions using combined data from three successive generations. We evaluated nine different genomic selection models, including three supervised machine learning architectures: gradient boosting, logistic regression, and random forest. Combining data across multiple generations did not, by itself, substantially improve the accuracy of most models, but the increased sample size provided genotyping confidence of loci with rare alleles. Correlation accuracy of all models significantly increased with the inclusion of low-frequency variants and strong-effect markers identified through a genome-wide association study. Gradient boosting machine learning models outperformed other genomic selection models across all training scenarios, suggesting enhanced capacity to learn generalizable genomic signals associated with dermo resistance. The best gradient boosting model achieved a peak correlation accuracy of 0.410, a substantial improvement over the previous peak accuracy of 0.274 from traditional models. Together, our results highlight the potential of machine learning for genomic selection and the significance of rare variants in determining dermo resistance.

Genomic signatures can provide key insight into the evolutionary history and remaining adaptive potential of threatened populations. As demographic decline erodes both diversity and the processes maintaining it, understanding how remaining variation is distributed becomes increasingly important for conserving species like the staghorn coral, Acropora cervicornis, a foundational but critically endangered Caribbean reef-builder. We analyzed 46 high-coverage A. cervicornis genomes from 10 locations across the tropical western Atlantic to evaluate neutral and adaptive structure, genomic diversity, demographic history, inbreeding, and connectivity, and generated a regional haplotype reference panel for future genomic monitoring. Genome-wide analyses recovered recurring regional substructure, but differentiation was modest and partly explained by isolation-by-distance and spatial variation in effective migration. Subpopulations had similar levels of genomic diversity, shared demographic history, and limited evidence of local adaptation. These patterns support interpreting sampled Caribbean populations as a single evolutionarily significant unit (ESU) containing multiple regional management units (MUs), rather than as deeply divergent evolutionary lineages. Despite substantial retained variation and low current inbreeding, estimated contemporary effective population size was small, suggesting an increased vulnerability to the effects of drift as demographic collapse continues, especially if structure is reinforced by isolated management. Together, our findings emphasize the urgent need for interventions that preserve and enhance genomic diversity, including risk-managed assisted gene flow. Supported by the haplotype reference panel developed here, these strategies will require coordinated efforts across regional entities to conserve and restore A. cervicornis as a jointly managed, single ESU.

The balance between local adaptation and gene flow is rarely quantified when populations are hierarchically differentiated across multiple spatial scales. We addressed this gap in Atlantic salmon (Salmo salar) at its southern range edge, using 1342 individuals from 21 rivers along the French Atlantic and Channel coasts with a 60k SNP array, and combining estimates of dispersal with genome-wide scans for adaptive differentiation across spatial scales. Population structure was hierarchical, resolving five genetic clusters with differentiation among groups five times higher than among populations within them. Assignment tests revealed a marked contrast between clusters, but unassigned individuals reached very high proportions within some clusters, indicating that gene flow has already blurred among-populations differences. Adaptive differentiation was detected among and within clusters, but the loci involved overlapped only partially across scales and among clusters, indicating that adaptation at each hierarchical level relies on distinct sets of variants. Dispersal was a poor guide to where this signal persisted: retaining putative dispersers reduced the candidate set more than threefold, showing that immigrant genotypes could dilute among-populations allele-frequency contrasts within a single generation. Yet differentiation among populations remained strong, as expected if dispersers reproduce poorly. Resolving the balance between local adaptation and gene flow therefore requires accounting for dispersal and adaptive differentiation at different spatial scales, with direct implications for conserving hierarchically structured populations at climatically vulnerable range edges.

Understanding how natural populations adapt to complex environmental gradients is crucial for predicting evolutionary responses to global change. The pine processionary moth (Thaumetopoea pityocampa), a major forest pest expanding northward and upward in Europe, provides an ideal model to explore genomic adaptation along altitudinal and/or latitudinal gradients. We combined pooled and individual whole- genome resequencing of four pairs of low- and high-elevation populations from Spain, Italy and France (mainland and Corsica) to detect signatures of local adaptation. Population structure analyses revealed strong differentiation among distant populations but limited divergence within altitude pairs. Genome scans identified a few candidate regions under selection, and we notably uncovered a large (~1 Mbp) region showing reduced nucleotide diversity, negative Tajima's D, and fixed allele differences between high- and low-altitude populations, consistent with a recent selective sweep. This region includes a cluster of cytochrome P450 genes and the voltage-gated sodium channel gene para, both involved in detoxification and insecticide resistance. Although signatures of selection were observed at this locus in two distant population pairs, no definitive evidence of recent introgression was found between these distant populations, suggesting independent evolution. Altogether, our results do not show clear evidence for altitudinal adaptation in T. pityocampa, but suggest that xenobiotic exposure could exert a strong selective pressure locally. Our study highlights candidate genes and regions for further investigation of resistance evolution in an insect species in the wild.

Perkinsus marinus is a protist that causes the wasting disease dermo in the Eastern oyster, Crassostrea virginica. The disease causes high mortality of both wild and cultured oysters, impacting ecosystem health and aquaculture. Despite decades of research, the genetic mechanisms of dermo resistance remain poorly understood. Traditional selective breeding based on phenotypes has yielded improvements in dermo resistance, but progress has been slow, likely due to the trait's complex genetic architecture. Understanding the genetic mechanisms underlying dermo resistance may facilitate advanced breeding to accelerate improvement. This study conducted genome-wide association studies (GWAS) of dermo resistance, leveraging a multi-generational dataset comprising 2,423 oysters phenotyped for survival following a dermo challenge and genotyped using a high-density 66K SNP array. A two-stage GWAS with more inclusive quality control identified 48 dermo-resistance markers, including many associated with genes for innate immune response and energy metabolism. Most dermo-resistance markers showed rare minor alleles occurring at higher frequencies in oysters that died after challenge, suggesting that these rare alleles are deleterious and associated with dermo susceptibility. The strongest association was with a polymorphism in the mucin-5AC-like gene, explaining 8.1% of phenotypic variation and suggesting that mucus production plays a crucial role in dermo resistance. The inclusion of these markers has improved genomic prediction accuracy, and their identification provides new insights into genomic variation and the architecture of dermo resistance, informing future strategies for genetic improvement.

Prolonged use of the few insecticide classes available for long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) has driven widespread physiological resistance of malaria vector mosquitoes to this limited arsenal of active ingredients. However, recent innovations like next-generation LLINs (NG-LLINs) containing two complementary insecticides and new insecticide classes for IRS offer new opportunities for pre-emptive resistance management by deploying more diversified actives as mixtures, combinations, rotations or mosaics. Here a deterministic model of mosquito foraging behaviour was formulated to predict the probabilities of deterrence, mortality or successful feeding across repeated feeding attempts in scenarios with different combinations of NG-LLINs and/or IRS micro-mosaics with varying levels of insecticide diversification between neighbouring houses. Final fates were classified based on whether or not the mosquito eventually died or successfully fed, and whether the latter occurred indoors or outdoors after exposure to zero, one or several IRS insecticides. The primary outcome was the probability that a single F mosquito carrying a novel resistance trait to a new IRS insecticide successfully feeds, survives and reproduces, thereby establishing those traits within the population. The secondary outcome was the selection coefficient governing the spread of such novel resistance traits from the F generation onwards. For highly anthropophagic and endophagic vectors like Anopheles funestus, combining NG-LLINs with IRS micro-mosaics using two insecticides may reduce emergence rates for novel resistance traits against IRS insecticides by approximately 2 to 2.5-fold, mainly through direct killing by NG-LLINs, although exposure to both IRS actives when forced to visit multiple houses also contributes to a lesser extent. However, such resistance management benefits are fundamentally constrained by outdoor feeding behaviours that limit or completely prevent indoor insecticide exposure. Increasing IRS micro-mosaic insecticide diversity beyond two actives is unlikely to further dampen resistance emergence rates because few mosquitoes survive long enough without feeding to encounter several IRS treatments. Once a resistance trait becomes established in the vector population, selection coefficients remain consistently high enough to force the spread of those traits, regardless of intervention combination. For more exophagic, zoophagic vectors like An. arabiensis, NG-LLINs plus IRS micro-mosaics are not expected to provide any meaningful resistance management benefit because frequent outdoor feeding, often on animals, allows them to largely avoid insecticide exposure altogether. Exclusively indoor-focused vector control strategies may not satisfactorily slow insecticide resistance emergence and spread, so new outdoor protection measures that close these coverage gaps with complementary insecticides will be needed.

The Atlantic surfclam, Spisula solidissima solidissima supports multimillion dollar harvests in the western North Atlantic, including Georges Bank and Mid-Atlantic Bight populations along the continental shelf. Surfclam populations in this part of the Exclusive Economic Zone (3-200 nm from shore) are managed as a single unit, but demographic connectivity has never been tested directly. This study analyzed >8,500 SNPs in 548 Atlantic surfclams sampled across the USA harvested range to infer population structure, genomic diversity, and gene flow patterns. Genomic analyses included eight sampled and 10 reference Spisula solidissima similis samples, a morphologically cryptic nominal subspecies previously known only from nearshore habitats. Novel morphologically cryptic population structure was identified in S.s. solidissima. One operational taxonomic unit (OTU-A) was found only in the inshore region of southern New England, south of Cape Cod. The other population unit, OTU-B, was found offshore and in Cape Cod Bay. The federal fishing grounds only had OTU-B clams. Populations within each OTU were connected by gene flow, justifying current fishery management practices. Nearshore state waters had mixed OTU stocks. Hybridization was analyzed between the two OTUs and between the two subspecies based on nuclear SNPs and asymmetrical mitochondrial DNA introgression. Finally, we demonstrated the utility of a novel SNP panel for diagnosing all three taxa and their hybrids.

European white oaks grow from the Mediterranean coast to Southern Scandinavia, a huge environmental gradient that has likely fostered environmental adaptation. In the face of climate change, leveraging adaptations through assisted gene flow could help improve drought tolerance and maintain forest health, but requires an understanding of the species-specific patterns of adaptation to be successful at the target location. In this study, three common gardens were established in Switzerland, Tuerkiye, and Austria for two European white oak species (Quercus robur, and Q. pubescens) using provenances from Central and Southern Europe. Almost 900 oak seedlings were measured at key water-use efficiency and life history traits for their first two year of life and genotyped with low coverage whole-genome sequencing. Trait heritability and environmental adaptation were then explored through pedigree-free animal models, while the genomic architecture of traits was mapped using a genome wide association study ("GWAS"). Across the species, the heritability of measured traits was moderate to high, but common garden had a strong impact, signalling an environmental effect on the phenotype. Adaptation to precipitation seasonality was detected in key productivity and growth traits for both species, but had a small effect on absolute trait values. The GWAS identified a striking 150 kbp association in the Cyclic Nucleotide-Gated Ion Channel gene family with leaf d13C values. This gene family is involved in stomata opening and likely impacts the intrinsic water use efficiency under stress. Together, the strong signals of phenotypic plasticity and rather weak signals of climatic adaptation in seedlings suggest that assisted gene flow in these two white oaks is relevant only for highly drought-sensitive populations, if conducted managers should focus on seeds sources with high precipitation seasonality and smaller leaf sizes.

Mosquito-borne pathogens cause approximately 700,000 deaths annually, highlighting the need for effective and sustainable vector control strategies. CRISPR-based gene drives offer a potential approach for suppressing disease-vector populations, but autonomous drives may spread beyond intended populations. Split gene drives, in which the Cas9 and guide RNA components are genetically separated, provide an inherently confinable alternative because biased inheritance depends on the co-occurrence of both components. Here, we develop the first split suppression gene drives in Ae. aegypti, targeting the female reproductive genes doublesex (dsx) and yellow-g (yg). The yg-targeting drive exhibited moderate super-Mendelian inheritance, reaching 73.4% transmission in the highest-performing cross, whereas the dsx-targeting drive exhibited little detectable inheritance bias. Conversely, disruption of dsx imposed substantially stronger reproductive fitness costs, including complete sterility in homozygous females, whereas yg disruption produced a less severe fertility phenotype. Mathematical modeling parameterized with these experimental data predicted that three of four split-drive configurations could achieve population suppression at lower release intensities or shorter release durations than pgSIT across portions of the modeled parameter space. Together, these results reveal that inheritance efficiency and reproductive load can impose competing constraints on split suppression drive performance: stronger suppression-associated fitness effects do not necessarily translate into improved drive performance when accompanied by limited drive propagation. Our findings establish the feasibility of split suppression gene drives in Ae. aegypti and demonstrate that effective suppression drive design requires balancing inheritance efficiency with reproductive fitness costs rather than maximizing either independently.

Risk tolerance is influenced by both environmental and genetic factors and may be shaped by local selective pressures in hazardous environments. In Northern Senegal, fishermen from the village of Guet Ndar are chronically exposed to high occupational mortality risk, making this population a potential model for local adaptation to risky environments. In a previous study, we showed that men from this fishing community were less risk-tolerant than men from a nearby farming village, although this difference could not be explained by variation at the DRD4 locus. Here, we investigated whether genetic variants previously associated with general risk tolerance in large genome-wide association studies (GWAS) exhibit signals of local adaptation in these Senegalese populations. We genotyped 44 candidate SNPs in 373 individuals sampled from the risky fishing area and the non-risky farming area. Population genetic analyses revealed extremely low and non-significant differentiation between the two populations (mean FST = 0.0003, p = 0.33), indicating that the two groups are genetically indistinguishable at these loci. Furthermore, none of the candidate SNPs showed a significant association with experimentally measured risk tolerance after correction for multiple testing. These results provide no evidence that local adaptation for risky behavior has occurred, several explanations may account for this negative result.

Tree populations can respond to climate change through migration, phenotypic plasticity, or genetic evolution. Despite long generation times of forest tree species, recent studies suggest that their evolutionary responses may occur rapidly. Using oaks as a model system, we synthesize evidence from 88 common garden studies and from historical, retrospective and longitudinal approaches to explore how populations have adapted to climatic variability across different biomes, and assess the consistency and pace of evolutionary responses across spatial and temporal climatic gradients. We found that approximately 61% of the studies exhibited significant differences among populations but climatic drivers and adaptive strategies differed among biomes. Temperature-related clines predominated in temperate regions, with populations from warmer origins showing longer growing seasons and higher growth potential. In seasonally dry biomes, aridity favored increased drought tolerance in Mediterranean populations but drought avoidance in tropical populations. Allochronic studies revealed genetic changes over decades to millenia in response to climate changes, with warming associated with increased growth and reduced specific leaf area in temperate oaks. Thus, spatial differentiation and temporal evolution were generally congruent in direction for most traits except for leaf unfolding, while short-term evolutionary rates exceeded long-term estimates by two to three orders of magnitude. In summary, provenance trials can provide useful information on the direction of climate-driven evolution for some traits, but may underestimate its contemporary pace. More studies are needed to evaluate whether standing genetic variation of forest tree species is sufficient to track current climate change.

Genomic data can assist conservation planning, but implementation of genetic-based recommendations is rare. Closing this "conservation genetics gap" is crucial for restoration and translocation decisions, particularly for highly fragmented ecosystems like pine barrens. We first analyze the conservation genetics gap in pine barren management organizations using a survey. We then analyze whole-genome data to characterize population structure and diversity in the pine barren keystone plant Lupinus perennis. We find little genetic difference among L. perennis populations in the Northeast United States. While populations show signs of isolation by distance (rho = 0.785, p = 0.001), we find no correlation between population size and heterozygosity (Estimate = 0.003, p = 0.357) or inbreeding (Estimate = 0.028, p = 0.416). We also find no effect of seed source population on forecasted effective population size in restoration simulations. These findings and our recommendation that managers consider non-local seed provenancing for L. perennis restorations in the Northeast have been integrated into SWAP policy in two states. We generally recommend practitioners record seed provenance, save genetic material for future testing, and incorporate fitness metrics into restoration monitoring. To effectively close the conservation genetics gap, researchers providing conservation recommendations must consult with practitioners before designing experiments.

Biodiversity losses continue to outpace traditional management, underscoring the need to understand adaptive capacity and the potential for interventions to increase fitness under climate change. We undertook a genome-wide association study on 156 Acropora palmata genets to investigate the genomic basis of areal growth, endosymbiont association, and thermal tolerance. Seven peaks on chromosomes 1, 3 and 14 were associated with endosymbiont shuffling and two peaks on chromosome 4 were associated with areal growth. As variants were located in non-coding regions we incorporated additional data from an independent field-transplant experiment to investigate their relationship with patterns of gene expression. Intersection of these datasets implicated melanocortin-like receptor activity and Ran GTPase activating protein 1 in endosymbiont composition and surface area growth, respectively. Results indicate that growth and endosymbiont associations may represent more viable intervention targets than temperature tolerance and highlight the need to better understand the role of non-coding variation in basic biology and development of restoration interventions.

Long breeding cycles constrain genetic gain in Scots pine, while mature progeny trials can provide reference populations for genomic selection. We combined phenotypic records from two German trials established in 1990 with dense SNP data. We complemented these data with an offspring-level parentage audit, duplicate filtering, and simulations of breeding strategies. Of 1,986 phenotype-matched genotyped trees, 1,668 formed the parentage-green set and 1,651 remained after exclusion of 17 near-duplicate samples. The final population comprised 1,521 supported controlled-cross offspring and 130 mother-known, father-unknown offspring from 87 progeny labels. Across 14 trait-by-age measurements, GBLUP gave the clearest improvements for diameter and volume from age 20 onwards, whereas height was more mixed. At age 35, ABLUP versus GBLUP heritability was 0.155 versus 0.235 for diameter, 0.265 versus 0.263 for height, and 0.172 versus 0.236 for volume. Leave-progeny-out predictive ability increased from 0.205 to 0.238, 0.249 to 0.265, and 0.203 to 0.239, respectively. SNPscan_breeder simulations compared phenotypic selection, progeny testing, cross-generation genomic selection, and genomic selection with phenotypic thinning under five diversity variants. Progeny testing produced the greatest cumulative gain, but its 45-year cycle reduced annual response. Under the base assumptions, genomic selection with phenotypic thinning gave the highest annual gains for height and fungal resistance, whereas pure genomic selection gave the highest annual diameter gain. Genomic strategies accumulated more kinship than conventional strategies, although a {lambda} = 0.10 kinship penalty improved founder retention with little loss of gain; no mitigation option simultaneously maximised gain, prediction accuracy, and diversity. These results support genomic shortlisting within a field-tested programme with explicit reference-population updating and diversity management.

Ecological heterogeneity within a species native range can shape genomic variation available for subsequent range expansion and biological invasion. The cactus moth, Cactoblastis cactorum, is a South American oligophagous herbivore on Opuntia that has become an invasive pest outside its native range. In Argentina, populations span wide climatic and geographic gradients and exploit both native Opuntia species and the introduced crop O. ficus-indica. Using ddRADseq data from 136 individuals across 28 populations, we combined genome-wide differentiation scans (XtX), a host-use contrast, and genotype-environment association (GEA) analyses to identify candidate genomic signatures associated with contemporary ecological variation. XtX analyses detected 12 candidate regions, whereas the host-use contrast identified 17 regions differentiating populations sampled from O. ficus-indica and native Opuntia, several of which persisted in a geographically restricted sensitivity analysis. GEA analyses identified 167 covariate-specific loci, including candidate genes associated with detoxification and metabolism (CYP6B2), oxygen-sensing pathways (Egln1), and circadian regulation (TIMELESS). Convergence among analyses was limited but stronger than expected by chance. Temperature-associated loci in fax and Bag6 occurred within an XtX candidate region, whereas Hspg2 was independently recovered in host-use and environmental analyses and retained after geographic restriction. Most candidate variants occurred in non-coding genomic contexts, protein-altering variants were uncommon, and candidate genes spanned diverse functions, consistent with a potentially regulatory and polygenic architecture of adaptation. Overall, our results reveal a heterogeneous genomic landscape associated with climatic variation and introduced-host use, with recurrent loci providing the strongest evidence for localized adaptive differentiation across the native range of C. cactorum.

In Germany, insecticide use is recommended during mosquito-borne disease outbreaks, yet nationwide data on insecticide resistance in key vector species are scarce. This is particularly relevant for Culex pipiens s.l., a vector of West Nile virus. Here, we provide the first broad-scale assessment of molecular insecticide resistance (kdr and ace-1 mutations) in Cx. pipiens f. pipiens across Germany. We associated insecticide resistances with environmental conditions, potential links with mitochondrial variation of the cytochrome c oxidase I (COI) gene as well as phenotypic resistance (CDC bottle assay with permethrin) of the Frankfurt population with elevated kdr frequencies. Kdr resistance alleles were detected in German Cx. pipiens f. pipiens reaching frequencies of up to 50% in the metropolitan areas of Berlin and Frankfurt am Main. Annual precipitation was negatively associated with kdr allele frequency. Interestingly, ace-1 resistance mutations were only present in the metropolitan area of Frankfurt am Main and in Muggensturm (Rhine valley, southwestern Germany). COI haplotype analyses revealed low genetic variability within German Cx. pipiens f. pipiens populations with no association to the insecticide resistance genotype. Phenotypic assays of the Frankfurt population showed early signs of permethrin resistance, but unmatched genotypic and phenotypic resistance state. This study provides the first broad-scale assessment of molecular insecticide resistance in Cx. pipiens f. pipiens across Germany, establishing a baseline for resistance surveillance and vector control. Further research is needed to identify factors shaping phenotypic resistance, as molecular markers alone may not reliably predict resistance. Since sampling was restricted to urban green spaces, broader sampling across environmental gradients is needed to identify additional environmental effectors.