Conserved numts mask a highly divergent mitochondrial- <i>COI</i> gene in a species complex of Australian stingless bees <i>Tetragonula</i> (Hymenoptera: Apidae)

<i>Tetragonula carbonaria</i>, <i>Tetragonula davenporti, Tetragonula hockingsi</i> and <i>Tetragonula mellipes</i> comprise a species complex of Australian stingless bee species known as the 'Carbonaria' group. The species are difficult to distinguish morphologically and the major species-defining characters relate to comb architecture and nest entrance ornamentation. The taxonomy of the group is further complicated by likely nuclear mitochondrial pseudogenes (numts) and inter-specific hybrids. Here we demonstrate the existence of <i>COI</i> numts and isolate and characterize the 'true' mt-<i>COI</i> gene in <i>T. carbonaria</i> and <i>T. hockingsi</i>. Numts were isolated from enriched-nuclear DNA extraction followed by PCR amplification and Sanger sequencing, and were recognized by the presence of deletions and/or premature stop codons in the translated sequences. The mt-<i>COI</i> sequences were obtained from NGS sequencing using purified mtDNA. In <i>T. carbonaria,</i> two numts (numt1 and numt2) were identified and a third (numt3) was identified in <i>T. hockingsi</i>. Numt2 and numt3 are similar (1.2% sequence divergence), indicating a recent common origin. The genetic distance between the mt-<i>COI</i> of the two <i>Tetragonula</i> species was higher than might be expected for closely related species, 16.5%, corroborating previous studies in which <i>T. carbonaria</i> and <i>T. hockingsi</i> were regarded as separate species. The three numts are more similar to the <i>COI</i> of other stingless bee species, including Australian <i>Austroplebia australis</i> and South American <i>Melipona bicolor</i> (81.7-83.9%) than to the mt-<i>COI</i> of their own species (70-71.4%). This is because the mt-<i>COI</i> of <i>T. carbonaria</i> and <i>T. hockingsi</i> differ greatly from other Meliponinae. Our findings explain some formerly puzzling aspects of Carbonaria biogeography, and misinterpreted amplifications.