Abstract
Mitochondrial genomes serve as essential tools in evolutionary biology, phylogenetics, and population genetics due to their maternal inheritance, lack of recombination, and conserved structure. Traditional morphological methods for identifying nematodes are often insufficient for distinguishing cryptic species complexes. This study highlights recent advancements in nematode mitochondrial genome research, particularly the impact of long-read sequencing technologies such as Oxford Nanopore. These technologies have facilitated the assembly of mitochondrial genomes from mixed soil samples, overcoming challenges associated with designing specific primers for long PCR amplification across different groups of parasitic nematodes. In this study, we successfully recovered and assembled eleven nematode mitochondrial genomes using long-read sequencing, including those of two plant-parasitic nematode species. Notably, we detected Heterodera cruciferae in Victoria, expanding its known geographic range within Australia. Additionally, short-read sequencing data from a previous draft genome study revealed the presence of the mitochondrial genome of Heterodera filipjevi. Comparative analyses of Heterodera mitogenomes revealed conserved protein-coding genes essential for oxidative phosphorylation, as well as gene rearrangements and variations in transfer RNA placement, which may reflect adaptations to parasitic lifestyles. The consistently high A+T content and strand asymmetry observed across species align with trends reported in related genera. This study demonstrates the utility of long-read sequencing for identifying coexisting nematode species in agricultural fields, providing a rapid, accurate, and comprehensive alternative to traditional diagnostic methods. By incorporating non-target endemic species into public databases, this approach enhances biodiversity records and informs biosecurity strategies. These findings reinforce the potential of mitochondrial genomics to strengthen Australia's as well as the global biosecurity framework against plant-parasitic nematode threats.