Amborella trichopoda (Amborellaceae) is the single living sister species of all other extant flowering plants and only occurs in rain forest habitats on the remote island of New Caledonia. These features make Amborella an important species in which to study genetic variation, including gene presence/absence variants (PAVs). Here, we apply the reference genome based iterative mapping and assembly strategy (Bayer et al., 2020) to assess gene diversity across 10 diverse individuals. The N50 of the newly assembled contigs was 1.2 kb, indicating similar contiguity to those in comparable pan-genome studies (Brassica oleracea, 0.6–1.9 kb; Brassica napus, 1.2 kb; Solanum lycopersicum L., 1.4 kb; Musaceae, 0.7–2.4 kb; and Oryza sativaL., 1.1 kb)(Goliczet al., 2016a; Hurgobinet al., 2018; Wang et al., 2018; Gao et al., 2019; Rijzaani et al., 2021)(Supporting

Information Table S1). We identified 2765 additional genes not present in the reference assembly and found thatAmborellamay have relatively few dispensable genes (3136, 10.4%)(Table S2) compared with studies in other species (maize, 60.88%; Brassica oleracea, 18.71%; bread wheat, 42.30%; Brachypodium distachyon, 45%; and cultivated rice, 51.5%)(Hirsch et al., 2014; Golicz et al., 2016a;