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Current sequencing technologies are making available unprecedented amounts of genetic data for a large variety of species including nonmodel organisms. Although many phylogenomic surveys spend considerable time finding orthologs from the wealth of sequence data, these results do not transcend the original study and after being processed for specific phylogenetic purposes these orthologs do not become stable orthology hypotheses. We describe a procedure to detect and document the phylogenetic distribution of orthologs allowing researchers to use this information to guide selection of loci best suited to test specific evolutionary questions. At the core of this pipeline is a new phylogenetic orthology method that is neither affected by the position of the root nor requires explicit assignment of outgroups. We discuss the properties of this new orthology assessment method and exemplify its utility for phylogenomics using a small insects dataset. In addition, we exemplify the pipeline to identify and document stable orthologs for the group of orb-weaving spiders (Araneoidea) using RNAseq data. The scripts used in this study, along with sample files and additional documentation, are available at https://github.com/ballesterus/UPhO.

A New Orthology Assessment Method for Phylogenomic Data: Unrooted Phylogenetic Orthology