Improving the Resolution of Inter-Familial Relationships Within Palaeacanthocephala by Mitophylogenomics Including Isthmosacanthidae (Polymorphida) and Transvenidae (Echinorhynchida)

oxidase subunit I, and nuclear (nc) 18S rRNA and 28S rRNA genes provide limited resolution for deeper nodes. Mt genomes have clarified some relationships, but several families-including Transvenidae and Isthmosacanthidae-have lacked any mitogenomic representation. In this study, we sequenced complete mt genomes from eight palaeacanthocephalan species, including the first representatives of these two families, and evaluated the phylogenetic performance of four datasets: (1) amino acid sequences of mt protein-coding genes, (2) their nucleotide sequences, (3) mt 12S and 16S rRNA gene sequences combined with the data in (2), and (4) nc 18S and 28S rRNA genes combined with the data in (3). Maximum-likelihood and Bayesian analyses revealed that the last dataset (4) produced the strongest and most stable support, recovering all major lineages of Echinorhynchida with high confidence and clarifying relationships among several historically problematic families. In contrast, internal relationships within Polymorphida remained weakly supported across all datasets despite the first mitogenomic inclusion of Isthmosacanthidae. All analyses consistently supported the paraphyly of Echinorhynchida with respect to Polymorphida. Although the present data do not yet permit robust inference of morphological character evolution, the well-supported phylogenetic backbone established here provides a critical foundation for re-evaluating classical morphological traits and for future integrative studies that incorporate nc genomic and transcriptomic data. Our findings substantially advance the systematic framework of Palaeacanthocephala and highlight key targets for future phylogenomic and comparative evolutionary research.