Echinochloa spp. is one of the most invasive weeds in rice fields worldwide. Acetolactate synthase (ALS) and acetyl-CoA carboxylase (ACCase) inhibiting herbicides are two of the most widely used rice herbicides. However, overuse has led to the resistance evolution of Echinochloa spp. to penoxsulam (ALS-inhibitor) and cyhalofop-methyl (ACCase-inhibitor). In this work, 137 different Echinochloa spp. populations were collected in different rice fields in Extremadura (western Spain) where lack of control was detected. Target-site based resistance (by sequencing ALS and ACCase gene) and characterization of Echinochloa species at the molecular level (based on PCR-RFLP analyses) were carried out in those populations. Most of the populations studied (111 of 137) belong to the E. oryzicola/E. oryzoides group. Three-point mutations were identified in ALS genes: Pro197Ser, Pro197Thr, and Ser653Asn, the first being the most frequent substitution in resistant plants. In the ACCase gene, the Ile1781Leu substitution was found. In both ALS and ACCase sequencing, evidence of heterozygosity was also observed. To assess whether cross-resistance patterns differed between mutations, two populations belonging to the E. oryzicola/E. oryzoides group had its most frequent mutations (Pro197Ser, population ech3-14 and Ile1781Leu, population ech114-10) chosen to be carried out in a dose-response assay. It was confirmed that Pro197Ser conferred resistance to triazolopyrimidine, imidazolinone, sulfonylurea, and pyrimidinyl benzoate families. On the other hand, the Ile1781Leu change gave resistance to aryloxyphenoxypropionate and cyclohexanedione families. Of the authorized herbicides in rice in Spain, more that 80% belong to these families. It is therefore important that farmers carry out an integrated control system that combines both chemical and non-chemical tools.

Societal Impact Statement: Weedy plants are a major constraint on agricultural productivity. Weedy rice is a weed that invades rice fields worldwide and is responsible for reductions in rice yields. Studies to date have detected multiple independent weedy rice origins in different parts of the world. We investigated the origin of weedy rice in Spain and Portugal and found that it has evolved from a cultivated rice variety group grown locally. Iberian weeds carry mutations that reverse domesticated pericarp color to its ancestral red color. Our results imply that management strategies are needed to prevent the evolution of troublesome weeds from cultivated ancestors.Summary: Weedy rice, a damaging conspecific weed of cultivated rice, has arisen multiple times independently around the world. Understanding all weedy rice origins is necessary to create more effective weed management strategies. The origins of weedy rice in Spain and Portugal, where there are no native Oryza species, are unknown. In this study, we try to identify the likely ancestors of Iberian weedy rice and the mechanisms involved in the evolution of two weedy traits, seed shattering, and red pericarps. We used genotyping by sequencing to understand the origin of Iberian weedy rice and its relationship to other weedy, wild, and cultivated rice groups worldwide. We also genotyped candidate genes for shattering and pericarp color. We find that weedy rice in the Iberian Peninsula has primarily evolved through de‐domestication of temperate japonica cultivars, with minor origins from exotic weedy rice. Iberian weeds have evolved the capacity to shatter seeds via novel loci and have acquired red pericarps via compensatory mutations in the Rc domestication gene. Our results suggest the Iberian weeds have experienced selection at multiple locations in the genome to establish as weeds, likely targeting male fertility genes among other functions. Our characterization of Iberian weedy rice adds to the growing evidence that de‐domestication of cultivated rice varieties is the main source of weedy rice worldwide. Their evolutionary versatility explains why weedy rice continues to be one of the most problematic weeds of cultivated rice.