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Genetic innovation may be linked to evolutionary success, and indeed, the invasive round goby mitochondrial genome sequence carries two novel features not previously described in Benthophilinae. First, the round goby mitochondrial genome carries a rearrangement of the tRNA cluster Ile-Glu-Met. Second, the round goby mitochondrial genome features a 1250 bp non-coding sequence insertion downstream of the D-loop region. In this publication, we test where in the goby phylogeny the novel tRNA arrangement first arose and whether the sequence insertion is associated with invasive populations only or a genuine feature of the species. We sequence native and invasive populations in Europe and North America, and show that all round gobies carry the sequence insertion. By sequencing the tRNA cluster in selected Gobiidae, we show that the tRNA arrangement arose at the root of the Benthophilinae species radiation., Mitochondrial DNA Part B: Resources, 4 (1)