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Abstract Background Unresolved taxonomic classification and paraphyly pervade the flatworm class Monogenea: the class itself may be paraphyletic and split into Polyopisthocotylea and Monopisthocotylea; there are some indications that the monopisthocotylean order Dactylogyridea may also be paraphyletic; single-gene markers and some morphological traits indicate that the family Ancyrocephalidae is paraphyletic and intertwined with the family Dactylogyridae. Methods To attempt to study the relationships of Ancyrocephalidae and Monopisthocotylea using a phylogenetic marker with high resolution, we sequenced mitochondrial genomes of two fish ectoparasites from the family Dactylogyridae: Dactylogyrus simplex and Dactylogyrus tuba. We conducted phylogenetic analyses using three datasets and three methods. Datasets were ITS1 (nuclear) and nucleotide and amino acid sequences of almost complete mitogenomes of almost all available Monopisthocotylea mitogenomes. Methods were maximum likelihood (IQ-TREE), Bayesian inference (MrBayes) and CAT-GTR (PhyloBayes). Results Both mitogenomes exhibited the ancestral gene order for Neodermata, and both were compact, with few and small intergenic regions and many and large overlaps. Gene sequences were remarkably divergent for nominally congeneric species, with only trnI exhibiting an identity value > 80%. Both mitogenomes had exceptionally low A + T base content and AT skews. We found evidence of pervasive compositional heterogeneity in the dataset and indications that base composition biases cause phylogenetic artefacts. All six mitogenomic analyses produced unique topologies, but all nine analyses produced topologies that rendered Ancyrocephalidae deeply paraphyletic. Mitogenomic data consistently resolved the order Capsalidea as nested within the Dactylogyridea. Conclusions The analyses indicate that taxonomic revisions are needed for multiple Polyopisthocotylea lineages, from genera to orders. In combination with previous findings, these results offer conclusive evidence that Ancyrocephalidae is a paraphyletic taxon. The most parsimonious solution to resolve this is to create a catch-all Dactylogyridae sensu lato clade comprising the current Ancyrocephalidae, Ancylodiscoididae, Pseudodactylogyridae and Dactylogyridae families, but the revision needs to be confirmed by another marker with a sufficient resolution. Graphical Abstract

Abstract Background Diplozoidae are monogenean (Monogenea: Polyopisthocotylea) fish parasites characterised by a unique life history: two larvae permanently fuse into an X-shaped “Siamese” organism. Taxonomy and phylogeny of Diplozoidae and Polyopisthocotylea remain unresolved due to the unavailability of molecular markers with sufficiently high resolution. Mitogenomes may be a suitable candidate, but there are currently only 12 available for the Polyopisthocotylea (three for Diplozoidae). The only available study of diplozoid mitogenomes found unique base composition patterns and elevated evolution rates in comparison with other Monogenean mitogenomes. Methods To further explore their evolution and generate molecular data for evolutionary studies, we sequenced the complete mitogenomes of two Diplozoidae species, Paradiplozoon homoion and Paradiplozoon yarkandense, and conducted a number of comparative mitogenomic analyses with other polyopisthocotyleans. Results We found further evidence that mitogenomes of Diplozoidae evolve at a unique, elevated rate, which was reflected in their exceptionally long branches, large sizes, unique base composition, skews, and very low gene sequence similarity levels between the two newly sequenced species. They also exhibited remarkably large overlaps between some genes. Phylogenetic analysis of Polyopisthocotylea resolved all major taxa as monophyletic, and Mazocraeidea was split into two major clades: (Diplozoidae) + (all four remaining families: Diclidophoridae, Chauhaneidae, Mazocraeidae and Microcotylidae). It also provided further confirmation that the genus Paradiplozoon is paraphyletic and requires a taxonomic revision, so the two species may have to be renamed Indodiplozoon homoion and Diplozoon yarkandense comb. nov. Conclusions Although our findings indicate that mitogenomes may be a promising tool for resolving the phylogeny of Polyopisthocotylea, elevated evolutionary rates of Diplozoidae may cause phylogenetic artefacts, so future studies should pay caution to this problem. Furthermore, as the reason for their elevated evolution remains unknown, Diplozoidae are a remarkably interesting lineage for other types of evolutionary mitogenomic studies. Graphical Abstract