HiMAP: robust Phylogenomics from Highly Multiplexed Amplicon sequencing

High-throughput sequencing has fundamentally changed how molecular phylogenetic datasets are assembled, and phylogenomic datasets commonly contain 50-100-fold more loci than those generated using traditional Sanger-based approaches. Here, we demonstrate a new approach for building phylogenomic datasets using single tube, highly multiplexed amplicon sequencing, which we name HiMAP (Highly Multiplexed Amplicon-based Phylogenomics), and present bioinformatic pipelines for locus selection based on genomic and transcriptomic data resources and post-sequencing consensus calling and alignment. This method is inexpensive and amenable to sequencing a large number (hundreds) of taxa simultaneously, requires minimal hands-on time at the bench (150,000 bp concatenated alignment) contained >40,000 phylogenetically informative characters, and although some discordance was observed between analyses, it provided unparalleled resolution of many phylogenetic relationships in this group. Most notably, we found high support for the generic status of Zeugodacus and the sister relationship between Dacus and Zeugodacus. We discuss HiMAP, with regard to its molecular and bioinformatic strengths, and the insight the resulting dataset provides into relationships of this diverse insect group.