Flatworms (Platyhelminthes) are a phylum of major medical and veterinary importance, causing huge human and domestic animal mortality and morbidity (Collins, 2017). The vast majority of flatworm species belong to the major parasitic radiation Neodermata, which is also the most speciose clade of parasitic animals on the planet (Carlson et al., 2020). Comparative mitochondrial genomic studies have reported that Neodermata exhibit the most rapidly evolving mitochondrial DNA sequences among all bilaterian animals (Bernt et al., 2013, Jakovlić et al., 2023). Neodermata were traditionally divided into three classes: Monogenea, Cestoda, and Trematoda. However, Monogenea is almost certainly paraphyletic and split into two classes: Polyopisthocotylea and Monopisthocotylea (Justine, 1998, Mollaret et al., 2000, Zhang et al., 2019, Brabec et al., 2023, Caña-Bozada et al., 2023). Whereas mitogenomes are available for multiple major lineages of the remaining three classes, they have been characterised only for only one out of the four orders of Polyopisthocotylea: Mazocraeidea.

Contrary to the remaining Polyopisthocotylea, which are ectoparasites of fishes, members of the polyopisthocotylean order Polystomatidea parasitise tetrapods, and many of them are endoparasitic (Sinnappah et al., 2001, Leeming et al., 2022). The origin of this ancient lineage may have coincided with the appearance of first aquatic tetrapods more than 400 million years ago (Verneau et al., 2002). Recent findings suggest two independent origins of obligatory endoparasitic lifestyle in Cestoda and Trematoda (Brabec et al., 2023, Caña-Bozada et al., 2023). Finally, another independent origin of endoparasitism in Neodermata is found in the monopisthocotylean genus Enterogyrus (Ancyrocephalidae), parasitising the digestive system (stomach) of fish hosts (Paperna, 1963). There is statistical evidence that mitogenomes of endoparasites exhibit elevated evolutionary rates compared with ectoparasitic lineages, possibly due to a decreased locomotory capacity, increased metabolic dependence on the host, reduced effective population size (Ne), or some other parameter (Jakovlić et al., 2023). On this basis, we hypothesised that this major life-history transition was accompanied by a shift in evolutionary pressures acting on the mitogenomes of Polystomatidea compared with other Polyopisthocotylea, and that endoparasitic neodermatans exhibit elevated evolutionary rates compared with ectoparasitic neodermatans. However, at the time of this study (July 2023), only one Polystomatidea mitogenome was available in GenBank: Diplorchis hangzhouensis (JQ038227). As this mitogenome remains non-characterised and unpublished at the time of this study, it remains unknown whether Polystomatidea exhibit any unique mitogenomic evolutionary features.

In addition, we noticed that there are indications of a strong mitonuclear discordance between the mitogenomic sequence data (mtDNA) and the nuclear genomic sequence data (nDNA) with regard to the topology of the four classes of Neodermata (Zhang et al., 2019, Brabec et al., 2023, Caña-Bozada et al., 2023). Mitonuclear discordance presents a good opportunity to test the strength of mitonuclear co-evolutionary pressures: due to the need for efficient functioning of the oxidative phosphorylation (OXPHOS) cycle, mitochondria-associated nuclear genome-encoded genes (MANE) are expected to exhibit correlated evolutionary rates with mitochondrial genes (Hill, 2015, Yan et al., 2019). On this basis, we hypothesised that this putative mitonuclear discordance, if confirmed, should be attenuated or even completely disappear when only MANE genes are analysed. To improve the data availability for underrepresented Polyopisthocotylea (Polystomatidea), we sequenced the mitogenome and transcriptome of Diplorchis sp. (most likely D. nigromaculatus) and assembled the mitogenome of Protopolystoma xenopodis from previously sequenced genomic data. We combined these sequences with data available in public databases to conduct comparative and evolutionary analyses using both mitochondrial and nuclear genomic data.

Therefore, the objectives of this study were: i) Conduct mitochondrial and nuclear phylogenomic analyses of Neodermata to confirm topological mitonuclear discordance; ii) attempt to identify underlying reasons for these putative conflicting phylogenetic signals by comparing evolutionary rates among the four classes of Neodermata and analysing selection pressures in mtDNA and nDNA sequences; iii) test the hypothesis of mitonuclear coevolution by assessing whether MANE genes produce an evolutionary pattern similar to mtDNA; iv) conduct comparative mitogenomic analyses to identify unique architectural and evolutionary features of so far uncharacterised Polystomatidea in comparison with other Polyopisthocotylea; v) conduct selection analyses aimed at identifying differences in mitogenomic evolutionary patterns between endoparasitic and ectoparasitic neodermatan lineages.