Different Phylogenomic Methods Support Monophyly of Enigmatic Mesozoa

Dicyemids and orthonectids were traditionally classified in a group called Mesozoa, but their placement in a single clade has been contested and their position(s) within Metazoa is uncertain. Here, we assembled a comprehensive matrix of Lophotrochozoa (Metazoa) and investigated the position of Dicyemida (= Rhombozoa) and Orthonectida, employing multiple phylogenomic approaches. We sequenced seven new transcriptomes and one draft genome from dicyemids (Dicyema, Dicyemennea) and two transcriptomes from orthonectids (Rhopalura). Using these and published data, we assembled and analyzed contamination-filtered datasets with up to 987 genes. Our results recover Mesozoa monophyletic and as a close relative of Platyhelminthes or Gnathifera. Because of the tendency of the long-branch mesozoans to group with other long-branch taxa in our analyses, we explored the impact of approaches purported to help alleviate long branch attraction (e.g., taxon removal, coalescent inference, gene targeting). None of these were able to break the association of Orthonectida with Dicyemida in the maximum likelihood trees. Contrastingly, the Bayesian analysis and site-specific frequency model in maximum likelihood did not recover a monophyletic Mesozoa (but only when using a specific 50 gene matrix). The classic hypothesis on monophyletic Mesozoa is possibly reborn and should be further tested.