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Abstract

The recent appearance of agricultural and glyphosate-resistant waterhemp populations in Canada provides an opportunity to investigate evolutionary questions about the genetics of adaptation. In this study, we used a population genetics-based approach to examine the routes by which glyphosate resistance evolved in Canada waterhemp populations. We first assembled a high-quality reference waterhemp genome using 87x genome coverage of Pacific Biosciences long-read data from a single waterhemp plant. The reference genome contained 663 Mb assembled into 2,514 contigs with an N50 of 1.7 Mb. A chromosome-level assembly was obtained by scaffolding the contigs onto the Amaranthus hypochondriacus genome. Population genetics analysis was then performed using whole-genome resequencing data from 173 waterhemp plants sampled from agricultural field populations in the United States (Illinois and Missouri) and Canada (Ontario) as well as natural (non-weedy) populations in Ontario. The analyzed data supported the existing paradigm of two ancestral lineages, A. tuberculatus var. rudis and A. tuberculatus var. tuberculatus. More specifically, var. rudis was found in Missouri, var. tuberculatus was prevalent in the non-weedy Canada populations, and var. rudis was prevalent in Illinois, but with increasing admixture with var. tuberculatus in eastern Illinois populations. Of the two glyphosate-resistant agricultural Canada populations, one was predominantly var. rudis and likely arose via migration from a weedy U.S. population. The other glyphosate-resistant Canada population, however, was predominantly var. tuberculatus and was not highly differentiated from the non-weedy Canada populations. Consequently, it appears that glyphosate-resistant waterhemp in Canada arose both from migration of preadapted genotypes and from independent evolution within a native population. Across all populations, EPSPS amplification accounted for much of the glyphosate resistance, being found in 71% of all glyphosate-resistant individuals. Analysis of the EPSPS amplified region suggested a single origin of EPSPS amplification in the var. tuberculatus glyphosate-resistant Canada population, but multiple origins among all the other glyphosate-resistant populations. In summary, this research indicates glyphosate resistance in Canada waterhemp populations has evolved via two independent pathways, and the EPSPS amplification event has occurred multiple times within this species. Additionally, the genomic resources generated will be valuable for further research on the genetics and evolution of an extremely dynamic weed.