Analysis of multiple‐herbicide resistant Amaranthus palmeri populations from Spain points to an introduction of the eccDNA from America

BACKGROUNDThe herbicide‐resistant invasive weed species Amaranthus palmeri threatens agricultural production and native plant ecology in Spain, as well as in other European countries. Understanding whether herbicide resistance alleles evolve in situ or are introduced via gene flow remains unclear. To address this, we characterized multiple resistance to acetolactate synthase (ALS)‐‐ and 5‐enolpyruvylshikimate‐3phosphate synthase (EPSPS)‐inhibiting herbicides in two Spanish A. palmeri populations at the plant level. Additionally, we analyzed the extra‐chromosomal circular DNA (eccDNA) to determine whether glyphosate resistance resulted from local selection pressure or was introduced by gene flow.RESULTSBoth populations exhibit individuals that survived both herbicide MoA, with multiple resistance mechanisms to ALS‐ and EPSPS‐inhibiting herbicides. Eight different ALS allele mutations were identified in resistant plants, including Pro‐197‐Ile, reported only in one species previously. Glyphosate resistance in the two populations is to the result of gene duplication mediated by eccDNA. Spanish and North American eccDNAs showed complete identity, with no single nucleotide polymorphisms (SNPs) found between the partial analyzed sequences of noncoding regions.CONCLUSIONWe confirm for the first time in Europe resistance to ALS and EPSPS inhibitors at both the population and individual levels in two Spanish A. palmeri populations. The absence of SNPs in the eccDNA from Spanish populations compared to the reference American sequence and the presence of target‐site mutations in the ALS gene occurred without selective pressure from ALS herbicides, suggests that the origin of resistance traits may have evolved elsewhere and been introduced from the place of origin to Spain. However, it is important to note that the limited number of populations studied and the partial sequencing of eccDNA do not provide definitive confirmation of the exact origins of resistance mechanisms. This work raises concerns about the arrival of this and potentially other new herbicide‐resistant A. palmeri populations in Europe posing challenges for management. © 2025 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.