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bioRxiv · 10.64898/2026.03.31.715258

Evolutionary persistence of a highly prevalent multicopy mitochondrial-derived nuclear insertion (Mega-NUMT) in Neotropical Drosophila flies

Montoliu-Nerin, M.·Strunov, A.·Heyworth, E.·Schneider, D. I.·Thoma, J.·Hua-Van, A.·Courret, C.·Klasson, L. J.·Miller, W. J.
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Abstract

Background: Although strict maternal transmission of mitochondria is a general feature of animals and humans for ensuring homogeneity in mitochondrial DNA (mtDNA) across generations, exceptions were reported in the recent past. For example, some extremely rare but spectacular cases of heteroplasmy and paternal transmission in humans have questioned the universal evolutionary principle. Hence, as an alternative, the Mega-NUMT concept was coined to explain this discovery and was thereafter partly proven to exist. This concept expands on the quite common transfer of mtDNA fragments to the nucleus (NUMTs) by considering the existence of multicopy mitochondrial nuclear insertions. Mega-NUMT reports are currently restricted to a few cases in animals, including humans. However, even in humans, their detailed genomic organization, natural prevalence, and potential biological functions remain unclear. Methodology/Principal Findings: Here, we discovered that up to 60 full-sized mitochondrial genomes are integrated into the nuclear genome of the neotropical fruit fly Drosophila paulistorum using long-read sequencing and confirmed their presence by in situ hybridization. The copies are organized in one cluster on chromosome 3, which we, due to its similarity with the Mega-NUMT concept, designated the Dpau Mega-NUMT. Contrary to the rarity in humans, this Mega-NUMT is found at high prevalence (40%) in both long-term laboratory lines and natural D. paulistorum populations of different semispecies. Additionally, the mitochondrial copies in the Mega-NUMT cluster are phylogenetically separated from the current mitotypes of D. paulistorum. Together, these observations suggest long-term maintenance of the Mega-NUMT in nature. Hence, we propose that the Dpau Mega-NUMT may have been transferred to the nuclear genome before D. paulistorum semispecies radiation and maintained at relatively high prevalence in nature by balancing selection due to yet undetermined functions. Conclusions/Significance: To our knowledge, this is the first verified existence and detailed dissection of a Mega-NUMT outside cats and humans. We show that Mega-NUMTs can be persistent in nature, even at high prevalence, potentially due to balancing selection. Our findings strengthen the importance of high-quality long-read sequencing technologies for deciphering complex repeat-rich genomic regions to deepen our understanding of the dynamics of genome evolution within genomic dark matter.

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Montoliu-Nerin, M., Strunov, A., Heyworth, E., Schneider, D. I., Thoma, J., Hua-Van, A., Courret, C., Klasson, L. J., Miller, W. J.. 2026-04-01. Evolutionary persistence of a highly prevalent multicopy mitochondrial-derived nuclear insertion (Mega-NUMT) in Neotropical Drosophila flies. https://doi.org/10.64898/2026.03.31.715258

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