A Phylogenomic Approach to Clarifying the Relationship of Mesodinium within the Ciliophora: A Case Study in the Complexity of Mixed-Species Transcriptome Analyses.

Recent high-throughput sequencing endeavors have yielded multigene/protein phylogenies that confidently resolve several inter- and intra-class relationships within the phylum Ciliophora. We leverage the massive sequencing efforts from the Marine Microbial Eukaryote Transcriptome Sequencing Project, other SRA submissions, and available genome data with our own sequencing efforts to determine the phylogenetic position of Mesodinium and to generate the most taxonomically rich phylogenomic ciliate tree to date. Regardless of the data mining strategy, the multiprotein data set, or the molecular models of evolution employed, we consistently recovered the same well-supported relationships among ciliate classes, confirming many of the higher-level relationships previously identified. Mesodinium always formed a monophyletic group with members of the Litostomatea, with mixotrophic species of Mesodinium-M. rubrum, M. major, and M. chamaeleon-being more closely related to each other than to the heterotrophic member, M. pulex. The well-supported position of Mesodinium as sister to other litostomes contrasts with previous molecular analyses including those from phylogenomic studies that exploited the same transcriptomic databases. These topological discrepancies illustrate the need for caution when mining mixed-species transcriptomes and indicate that identifying ciliate sequences among prey contamination-particularly for Mesodinium species where expression from stolen prey nuclei appears to dominate-requires thorough and iterative vetting with phylogenies that incorporate sequences from a large outgroup of prey.