Mitochondrial genome analyses uncover intriguing population structure across the invasive trajectory of an iconic invader

Invasive species offer insights into rapid adaptations in novel environments. The iconic cane toad ( Rhinella marina ) is an excellent model for studying rapid adaptation during invasion. Previous research using the mitochondrial NADH dehydrogenase 3 ( ND3 ) gene in the Hawai’ian and Australian invasive populations found a single haplotype, indicating an extreme genetic bottleneck following introduction. Nuclear genetic diversity also exhibited reductions across the genome in these two populations. Here, we investigated the mitochondrial genome diversity of cane toads across this invasion trajectory. We created the first reference mitochondrial genome of the cane toad with long-read sequencing and constructed a phylogeny of Anura full mitochondrial genomes. We used transcriptomic data of 125 individuals from the native (French Guiana) and introduced (Hawai’i and Australia) ranges to construct nearly-complete mitochondrial genomes for population genomics analyses. As expected, the cane toad belongs to family Bufonidae, distinct from genus Bufo . In agreement with previous investigations of these populations, we identified genetic bottlenecks in both Hawai’ian and Australian introduced populations, alongside evidence of population expansion in the invasive ranges. Although mitochondrial genetic diversity in introduced populations was reduced, our results revealed that it had been underestimated: we identified 45 mitochondrial haplotypes in Hawai’ian and Australian samples, none of which were found in the native range. Additionally, we identified two distinct groups of haplotypes from the native range, separated by a minimum of 110 base pairs (0.6%). These findings enhance our understanding of Anura phylogenetics and how invasion has shaped the genetic landscape of this species.