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 analysed 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.

Department of Biological Sciences The University of Alabama Campus Box 870344 Tuscaloosa AL 35487 USA

Department of Ecology Evolution and Marine Biology University of California Santa Barbara Santa Barbara CA 93106 USA

Department of Neuroscience and the Whitney Laboratory for Marine Bioscience University of Florida 9505 Ocean Shore Boulevard St Augustine FL 32080 USA

Department of Parasitology University of South Bohemia České Budějovice 37005 Czech Republic

Department of Zoology Faculty of Science University of South Bohemia České Budějovice 37005 Czech Republic

Laboratory of Molecular Ecology and Evolution Institute of Parasitology Biology Centre CAS České Budějovice 37005 Czech Republic

Molecular Cellular and Biomedical Sciences University of New Hampshire Durham NH 03824 USA

The Centre for Marine Science University of North Carolina Wilmington 57000 Marvin K Moss Lane Wilmington NC 28409 USA

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Different phylogenomic methods support monophyly of enigmatic 'Mesozoa' (Dicyemida + Orthonectida, Lophotrochozoa)