# Characterization and Comparative Analyses of Nuclear Mitochondrial DNAs in Genomes of the Leaf-Roller Moths (Lepidoptera: Tortricidae)

## TL;DR

This study identifies and analyzes nuclear mitochondrial DNA sequences (Numts) in leaf-roller moths, revealing their distribution and potential impact on molecular research.

## Contribution

The first systematic characterization of Numts in Tortricidae moths, revealing their genomic distribution and implications for molecular studies.

## Key findings

- All 27 Tortricidae species analyzed contain 9–208 Numts, with copy numbers correlating to nuclear genome length.
- Numts prefer AT-rich insertion sites and are predominantly derived from the mitochondrial cox1 gene.
- Numts pose risks for cox1-based DNA barcoding and phylogenetic studies due to co-amplification potential.

## Abstract

During eukaryotic evolution, mitochondrial DNA (mtDNA) fragments form Numts by integrating into nuclear genomes. Tortricidae lacked systematic Numt characterization, impeding molecular research. We analyzed Numts in 27 species (two subfamilies) with identification thresholds (E-value 10−4, >60% similarity, ≥50 bp). All species had 9–208 Numts, with counts correlating to nuclear genome length; Numts favored AT-rich insertion sites and derived mainly from cox1, highlighting risks for cox1-based molecular studies. This study systematically characterized Tortricidae Numt traits, informing molecular systematics and pest management for Tortricidae.

During eukaryotes evolution, mitochondrial DNA (mtDNA) fragments integrate into nuclear genomes, forming nuclear mitochondrial DNA sequences (Numts). Tortricidae (Lepidoptera), a species-rich and economically critical family, lacks systematic characterization of Numts, which hinders reliable molecular research. Here, we systematically characterized Numts in 27 Tortricidae species spanning two subfamilies via genome download, mitochondrial genome annotation, and Numt identification and characterization. With each species’ mtDNA as query, Numt identification was performed with an E-value threshold of 10−4 and a sequence similarity cut-off of >60%, with a minimum length of 50 bp to exclude spurious hits. Results showed that all species contained Numts, with copy numbers varying drastically (9–208). Numt numbers positively correlated with nuclear genome length, but not mitochondrial genome length. Numts insertion flanking regions had significantly higher AT content than nuclear genome, indicating the insertion preference for AT-rich regions. Numts were predominantly derived from the mitochondrial cox1 gene, highlighting the risk of co-amplification when cox1 is used as a DNA barcode for species identification or phylogenetic studies. This study represents a systematic charaterizition of copy number, length distribution, insertion sequence preferences, and mitochondrial gene origins of Numts in Tortricidae, offering valuable references for refining molecular systematics, comparative genomics, and pest management in Tortricidae and related lepidopteran groups.