• The adaptation of weeds to herbicide is both a significant problem in agriculture and a model of rapid adaptation. However, significant gaps remain in our knowledge of resistance controlled by many loci and the evolutionary factors that influence the maintenance of resistance.
• Here, using herbicide-resistant populations of the common morning glory (Ipomoea purpurea), we perform a multi-level analysis of the genome and transcriptome to uncover putative loci involved in nontarget-site herbicide resistance (NTSR) and to examine evolutionary forces underlying the maintenance of resistance in natural populations.
• We found loci involved in herbicide detoxification and stress sensing to be under selection and confirmed that detoxification is responsible for glyphosate resistance using a functional assay. We identified interchromosomal linkage disequilibrium (ILD) among loci under selection reflecting either historical processes or additive effects leading to the resistance phenotype. We further identified potential fitness cost loci that were strongly linked to resistance alleles, indicating the role of genetic hitchhiking in maintaining the cost.
• Overall, our work suggests that NTSR glyphosate resistance in I. purpurea is conferred by multiple genes which are potentially maintained through generations via ILD and that the fitness cost associated with resistance in this species is likely a by-product of genetic-hitchhiking.

On Jan 10, 2022, we planted 215 replicate seeds from multiple resistant and susceptible lineages per population (Table S10) in Cone-Tainers (Stewe and Sons) in a controlled growth room. After 30 days we subjected plants to four treatments: malathion (1 kg ai/ha OrthoMAX, Scotts Co.), glyphosate (3.4 kg ai/ha RoundUp PowerMax, Bayer), glyphosate and malathion, or a control. Prior to this experiment, we exposed 20 plants to five rates of malathion (N = 100) and identified 1 kg ai/ha as a rate that did not influence I. purpurea biomass or cause damage on its own. Malathion was applied using a handheld CO2 sprayer, and glyphosate was applied 1 hour later, again using a hand-held CO2 sprayer (Spraying Systems Co., Wheaton, IL) calibrated to deliver 187 L/ha. Twenty-four days post-treatment, we recorded death, harvested and dried plants for 3 days at 70C, and weighed each sample for an estimate of biomass.

To compare death and biomass between resistant and susceptible plants, we used log-likelihood tests and generalized linear models. Biomass was log-transformed (transformTukey function; Rcompanion v.2.0.0; Mangiafico, 2015) and used as the dependent variable with population type (R/S) and treatment as independent variables (biomass ~ population type + treatment). We assessed significance using the Anova function of the car package v.3.0.10 (Fox & Weisberg, 2018), and performed a pairwise comparison between groups using the lsmeans function from package lsmeans v2.30.0 (Lenth, 2016), adjusted for multiple tests using Tukey correction. Using the same general model, we also compared biomass between treatments for each population type (R vs S). To control for inherent differences in plant size across lineages, we standardized the biomass of each individual grown in the treatment environment by dividing biomass by the average of plants from the same maternal line grown in the control environment.