Smooth pigweed (Amaranthus hybridus) and unresolved Amaranthus spp. from Brazil resistant to glyphosate exhibit the EPSPS TAP-IVS substitution

The presence of glyphosate-resistant smooth pigweed (Amaranthus hybridus L.) biotypes has increased in southern Brazil in the last years, presenting the triple amino acid substitution TAP-IVS in the 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS), as previously found in Argentina. Some of these biotypes have morphological characteristics of smooth pigweed and redroot amaranth (Amaranthus retroflexus L.). The present study aimed to identify, through molecular markers, the herbicide-resistant species of Amaranthus from Brazil that have the TAP-IVS substitution and to analyze the occurrence of pollen-mediated gene flow (PMGF) as the source of TAP-IVS substitution in these biotypes. Six biotypes were evaluated by the internal transcribed spacer (ITS) sequences, of which two (AMACHY-S and CAMAQ-R) were molecularly classified as A. hybridus, and four (AMACRET-S, AMACVI-S, ARRGR-R, and SAOJER-R) were unclassified. Interestingly, all the glyphosate-resistant biotypes (ARRGR-R, SAOJER-R, and CAMAQ-R) had the TAP-IVS substitution, and an increase in EPSPS relative copy number; however, only CAMAQ-R was confirmed as A. hybridus. Although the biotypes ARRGR-R and SAOJER-R are closely related to A. hybridus and green pigweed (Amaranthus powellii S. Watson), their species identity could not be resolved. The biotype SAOJER-R also was resistant to ALS-inhibiting herbicides due to a tryptophan to leucine substitution at position 574 in acetolactate synthase (ALS). The evaluation of 119,746 seedlings in an intraspecific hybridization study of A. hybridus indicated an outcrossing frequency of 0.09%. In contrast, an absence of interspecific hybridization (A. hybridus × unclassified biotype – AMACVI-S) was found after screening 111,429 offspring. Unclassified biotypes might be derived from one or more ancient hybridization events and subsequently evolved the triple mutation independently. Alternatively, such biotypes could have evolved from recent hybridization events, which occur at a frequency below the level of detection in our study.