Phylogenetic Inference and Ancestral Character Reconstruction of Diphyllobothriid Tapeworms (Cestoda: Diphyllobothriidae).

Some groups of Diphyllobothriid tapeworms can cause foodborne or waterborne taeniasis, yet their systematics and evolution remain poorly understood. Based on mitochondrial genome data, this study reconstructed the phylogenetic trees of 11 cestode orders using maximum likelihood and Bayesian methods, clarified the valid species of Spirometra, traced the evolutionary history of four key ecological traits, and estimated the divergence times of major groups. Phylogenetic analyses supported Diphyllobothriidea and Bothriocephalidea as distinct clades, with Diphyllobothriidea forming a sister group to Haplobothriidea. In addition, three novel types of mitochondrial gene arrangements were identified in tapeworms, and variations in these arrangement types appear to correspond to changes in proglottid morphology. Species validation has shown that the available Spirometra mitogenomes represent only four valid species. Phylogenetic analysis identified two genetic lineages within S. mansoni, and a comparison of their representative sequences showed that the differences mainly lie in 12 PCGs, 7 tRNAs, 2 rRNAs, and 2 mNCRs. Ancestral character reconstruction suggested that tapeworms likely originated from freshwater fish hosts, with gradual adaptation to terrestrial hosts representing the main pathway of diversification, whereas transitions to marine environments were independent evolutionary events occurring in only a few groups. Molecular clock analyses indicated that tapeworm diversification began in the mid-to-late Oligocene, with episodic species radiations in different groups closely linked to host diversification and climatic changes. Notably, most groups within the family Diphyllobothriidae radiated during the Pleistocene, whereas Spirometra radiated during the Pliocene, coinciding with the origin of human-specialized cestodes. This study clarifies the phylogenetic position of diphyllobothriid tapeworms, infers the evolutionary patterns of ancestral traits across different tapeworm groups, and provides a theoretical basis for elucidating the origin and species diversity of tapeworms, as well as for assessing the transmission risk of pathogenic species.