16Wildfires can encourage the establishment of invasive plants by releasing potent germination 17 stimulants, such as karrikins. Seed germination of Brassica tournefortii, a noxious weed of 18 Mediterranean climates, is strongly stimulated by KAR1, which is the most abundant karrikin 19 produced from burning vegetation. In contrast, the closely-related yet non-fire-associated 20 ephemeral Arabidopsis thaliana responds preferentially to the less abundant KAR2. The α/β-21 hydrolase KARRIKIN INSENSITIVE2 (KAI2) is the putative karrikin receptor identified in 22Arabidopsis. Here we show that B. tournefortii differentially expresses three KAI2 23 homologues, and the most highly-expressed homologue is sufficient to confer enhanced 24 responses to KAR 1 relative to KAR 2 when expressed in Arabidopsis. We further identify two 25 variant amino acid residues near the KAI2 active site that explain the ligand selectivity. Our 26 results suggest that duplication and diversification of KAI2 proteins could confer upon weedy 27 ephemerals differential responses to chemical cues produced by environmental disturbance, 28 including fire. (146 words) 29 by the presence of a methyl group on the butenolide ring in KAR1, which is absent in KAR2 38 ( Supplementary Fig. 1a). Invasive plant species that are responsive to karrikins could utilise 39 natural and human-induced fires to facilitate their establishment 5, 6 . 40 Brassica tournefortii (Brassicaceae; Sahara mustard) is native to northern Africa and the Middle 41East, but is an invasive weed that blights many ecosystems with a Mediterranean climate and 42 chaparral-type vegetation that are prone to wildfires in North America, Australia and South 43 Africa. B. tournefortii seed can persist in the soil for many seasons, undergoing wet-dry cycling 44 that can influence dormancy and contribute to boom-bust cycles that outcompete native 45 3 species 7, 8 . B. tournefortii plants may radically alter fire frequency and intensity by influencing 46 fuel profiles 9, 10 , further exacerbating the impact of fire on susceptible native ecosystems. In 47 addition, seed of B. tournefortii is particularly responsive to smoke-derived karrikins, and 48 shows a positive germination response to KAR1 in the nanomolar range 11 . Accordingly, B. 49 tournefortii is particularly well positioned to invade areas disturbed by fire events 12, 13 . 50The putative karrikin receptor KARRIKIN INSENSITIVE 2 (KAI2) was identified in Arabidopsis 51 thaliana, a weedy ephemeral that originated in Eurasia but is now widely distributed 52 throughout the northern hemisphere 14, 15, 16 . Arabidopsis is not known to colonise fire-prone 53 habitats, but nevertheless seeds germinate in response to karrikins in the micromolar range 17 . 54Unlike most smoke-responsive species that respond more readily to KAR1 18, 19 , Arabidopsis 55 responds preferentially to the less abundant analogue KAR2 17 . KAI2 is an evolutionarily ancient 56 α/β-hydrolase and a paralogue of DWARF14 (D14), the receptor for strigolactones 20, 21 . 57 ...