The transition from a free-living lifestyle to endosymbiosis represents a large evolutionary shift, impacting various aspects of any organism’s biology, including its molecular basis. So far, it has been impossible to generalise the impact this lifestyle shift has on genomic architecture. This study explores this phenomenon using a new model system: neodalyellid flatworms (Rhabdocoela), a diverse assemblage of free-living and independently evolved endosymbiotic lineages. A uniquely comprehensive mitochondrial genomic dataset, consisting of 50 complete or partial mitogenome sequences (47 of which are new to science), is constructed, increasing the genomic resources available for rhabdocoel flatworms over tenfold. A robust phylogenomic framework is built, enabling an in-depth exploration of the molecular-genetic signatures associated with evolutionary shifts towards endosymbiosis. To understand speciation influenced by host phylogeny, first steps are taken to unravel the host-switching history of the largest endosymbiotic group of neodalyellids. We test several hypotheses regarding the potential consequences of a symbiotic lifestyle, and find heightened AT content, more pronounced AT skew, and relaxed selection on specific protein-coding genes in endosymbionts compared to their free-living counterparts. Numerous substitutions have accumulated in certain endosymbiotic lineages; however, the correlation with lifestyle remains uncertain. A high frequency of genetic rearrangements across all studied lineages is observed. Our findings affirm the variable nature of rhabdocoel mitogenomes and, for the first time, reveal distinct signatures of an endosymbiotic lifestyle in neodalyellid flatworms. This effort lays the groundwork for future research into the evolutionary and genomic consequences of a symbiotic lifestyle in this and other animal systems.

The Cyclophyllidea comprise the most species-rich order of tapeworms (Platyhelminthes, Cestoda) with, for helminths, perhaps the most severe health impact on wildlife, livestock, and humans. Rodent biodiversity of the Qinghai-Tibet Plateau (QTP) and its surrounding ranges provide a significant reservoir for numerous species of Cyclophyllidea. We collected cyclophyllidean species from QTP and Xinjiang province in China, resolving four unsequenced and likely new species. Phylogenetic construction of partial 18S rDNA, 28S rDNA and mitochondrial ( mt) genes provided high nodal support for the categorisation of the three of the putative new species, assigning each respectively to the genera Hydatigera (ex Eospalax fontainierii), Mesocestoides (ex Neodon irene) and Paranoplocephala (ex Neodon irene). Poor nodal support for the unidentified (‘new’) species collected from pika ( Ochotona curzoniae) in Yushu county, Qinghai province. Combined with the current investigation, the other three cyclophyllidean species found in this study ( Taenia caixuepengi, Taenia crassiceps and Versteria mustelae) may be widely distributed in western China. The phylogenetic reconstructions based on 28S rDNA and cox1- nad1 indicate that some families and genera may require taxonomic revision. Estimates of divergence time based on mt genes showed that the differentiation rate of tapeworms was strongly associated with the rate of change in the biogeographic scenarios caused by the uplift of the QTP, i.e. species differentiation of Cyclophyllidea was driven by host-parasite co-evolution caused by the uplift of QTP. We propose an “out of QTP” hypothesis for the radiation of cyclophyllideans.