Expanding the mitochondrial genomic toolkit for Polyneoptera: New mitogenomes and evaluation of reduced marker sets for phylogeny and DNA barcoding.

Polyneoptera comprises hemimetabolous insect orders of significant agricultural, ecological, and medical relevance, motivating phylogenetic and molecular research that has nevertheless focused predominantly on canonical mitochondrial markers. Here, we assembled new mitogenomes for Polyneoptera and evaluated the usefulness of genes located in nucleotide-diversity hotspots as markers for species identification and phylogenetic inference. To expand the available mitogenomic resources, raw sequencing data were retrieved from public databases, resulting in the assembly and annotation of 26 complete mitogenomes, all exhibiting the typical insect mitochondrial architecture. These newly assembled genomes were combined with publicly available mitogenomes from Orthoptera, Blattodea, Plecoptera, Mantodea, and Phasmatodea to reconstruct phylogenetic relationships using both complete and reduced datasets comprising nucleotide-diversity hotspot-associated genes. The performance of these hotspot regions was further assessed through barcoding gap analyses and comparisons with the most comprehensive datasets to identify candidate mitochondrial markers for molecular species identification and phylogenetic inference. Across orders, different mitochondrial regions, including the classical markers 16S and COX1, as well as genes from the NADH dehydrogenase complex, emerged as the most informative, although optimal markers varied among lineages. Overall, our findings highlight the value of publicly accessible sequencing data for generating high-quality genomic resources and improving phylogenetic and taxonomic tools.