Tetragonula carbonaria, Tetragonula davenporti, Tetragonula hockingsi and Tetragonula mellipes 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 COI numts and isolate and characterize the ‘true’ mt-COI gene in T. carbonaria and T. hockingsi. 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-COI sequences were obtained from NGS sequencing using purified mtDNA. In T. carbonaria, two numts (numt1 and numt2) were identified and a third (numt3) was identified in T. hockingsi. Numt2 and numt3 are similar (1.2% sequence divergence), indicating a recent common origin. The genetic distance between the mt-COI of the two Tetragonula species was higher than might be expected for closely related species, 16.5%, corroborating previous studies in which T. carbonaria and T. hockingsi were regarded as separate species. The three numts are more similar to the COI of other stingless bee species, including Australian Austroplebia australis and South American Melipona bicolor (81.7–83.9%) than to the mt-COI of their own species (70–71.4%). This is because the mt-COI of T. carbonaria and T. hockingsi differ greatly from other Meliponinae. Our findings explain some formerly puzzling aspects of Carbonaria biogeography, and misinterpreted amplifications.

Tetragonula carbonaria，Tetragonula davenporti，Tetragonula hockingsi和Tetragonula mellipes包括物种复杂称为“Carbonaria”组澳大利亚无刺蜂种。该物种很难在形态上区分，并且主要的物种定义特征与梳子的结构和巢口的装饰有关。该组的分类学由于可能的核线粒体假基因（数字）和种间杂种而更加复杂。在这里，我们证明了COI nums的存在，并分离和鉴定了T. carbonaria和T. hockingsi中“真正的” mt- COI基因。从富集核DNA提取物中分离核苷酸，然后进行PCR扩增和Sanger测序，并通过翻译序列中缺失和/或过早终止密码子的存在来识别。使用纯化的mtDNA从NGS测序获得mt- COI序列。在T. carbonaria中，鉴定出两个胡桃木（numt1和numt2），在T. hockingsi中鉴定出第三个（numt3）。Numt2和numt3相似（1.2％序列差异），表明是最近的共同起源。两个四角藻物种的mt- COI之间的遗传距离比紧密相关物种的预期遗传距离高16.5％，这证实了以前的研究。和T. hockingsi被认为是单独的物种。这三个数字与其他无刺蜂物种的COI更为相似，包括澳大利亚Austroplebia australis和南美双色Melipona bicolor（占81.7-83.9％），而不是其自身物种的mt- COI（占70-71.4％）。这是因为，MT- COI的T. carbonaria和T. hockingsi从其他Meliponinae差距很大。我们的发现解释了Carbonaria生物地理学的一些令人费解的方面，并错误地解释了放大现象。