Phylogenomic analyses of hundreds of protein-coding genes aimed at resolving phylogenetic relationships is now a common practice. However, no software currently exists that includes tools for dataset construction and subsequent analysis with diverse validation strategies to assess robustness. Furthermore, there are no publicly available high-quality curated databases designed to assess deep (>100 million years) relationships in the tree of eukaryotes. To address these issues, we developed an easy-to-use software package, PhyloFisher (https://github.com/TheBrownLab/PhyloFisher), written in Python 3. PhyloFisher includes a manually curated database of 240 protein-coding genes from 304 eukaryotic taxa covering known eukaryotic diversity, a novel tool for ortholog selection, and utilities that will perform diverse analyses required by state-of-the-art phylogenomic investigations. Through phylogenetic reconstructions of the tree of eukaryotes and of the Saccharomycetaceae clade of budding yeasts, we demonstrate the utility of the PhyloFisher workflow and the provided starting database to address phylogenetic questions across a large range of evolutionary time points for diverse groups of organisms. We also demonstrate that undetected paralogy can remain in phylogenomic “single-copy orthogroup” datasets constructed using widely accepted methods such as all vs. all BLAST searches followed by Markov Cluster Algorithm (MCL) clustering and application of automated tree pruning algorithms. Finally, we show how the PhyloFisher workflow helps detect inadvertent paralog inclusions, allowing the user to make more informed decisions regarding orthology assignments, leading to a more accurate final dataset.

Highlights d Discovery and cultivation of two new classes of marine ciliates thriving in anoxia d Phylogenomics reveals a major clade of obligate anaerobes in ciliates d Novel insights into evolution of mitochondrial metabolism in anaerobic eukaryotes d Transitions to obligate anaerobiosis might be facilitated by prokaryotic symbionts

Anaeramoebae are a divergent lineage of eukaryotes that shed light on the transition from anaerobic mitochondria to hydrogenosomes Highlights d Deep transcriptomic sequencing of two new anaerobic metamonads d The Anaeramoebae represents a new principal lineage of Metamonada d Anaeramoebae are free-living relatives of the parasitic parabasalids