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.

Department of Biochemistry and Molecular Biology Dalhousie University Halifax Canada

Department of Biological Sciences Mississippi State University Mississippi State Mississippi United States of America

Department of Biological Sciences Vanderbilt University Nashville Tennessee United States of America

Department of Biology and Ecology Faculty of Science University of Ostrava Ostrava Czech Republic

Department of Organismal Biology Uppsala University Uppsala Sweden

Faculty of Science University of South Bohemia České Budějovice Czech Republic

Institute for Genomics Biocomputing and Biotechnology Mississippi State University Mississippi State Mississippi United States of America

Institute of Parasitology Biology Centre Czech Academy of Sciences České Budějovice Czech Republic

Leibniz Institute of Freshwater Ecology and Inland Fisheries Ecosystem Research Berlin Germany

Science for Life Laboratory Uppsala University Uppsala Sweden

State Key Laboratory of Rice Biology and Ministry of Agriculture Key Lab of Molecular Biology of Crop Pathogens and Insects Institute of Insect Sciences Zhejiang University Hangzhou China

Unité d'Ecologie Systématique et Evolution CNRS Université Paris Saclay Paris France

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Mapping the metagenomic diversity of the multi-kingdom glacier-fed stream microbiome

Reconstructing the last common ancestor of all eukaryotes

Expanded gene and taxon sampling of diplomonads shows multiple switches to parasitic and free-living lifestyle

Comparative genomics of Ascetosporea gives new insight into the evolutionary basis for animal parasitism in Rhizaria

New plastids, old proteins: repeated endosymbiotic acquisitions in kareniacean dinoflagellates

Encyclopedia of Family A DNA Polymerases Localized in Organelles: Evolutionary Contribution of Bacteria Including the Proto-Mitochondrion

Mitochondrial genomes revisited: why do different lineages retain different genes?

Create, Analyze, and Visualize Phylogenomic Datasets Using PhyloFisher

Genomics of Preaxostyla Flagellates Illuminates the Path Towards the Loss of Mitochondria

Lessons from the deep: mechanisms behind diversification of eukaryotic protein complexes

A Novel Group of Dynamin-Related Proteins Shared by Eukaryotes and Giant Viruses Is Able to Remodel Mitochondria From Within the Matrix

Reconstruction of Plastid Proteomes of Apicomplexans and Close Relatives Reveals the Major Evolutionary Outcomes of Cryptic Plastids

Evidence for an Independent Hydrogenosome-to-Mitosome Transition in the CL3 Lineage of Fornicates

An Enigmatic Stramenopile Sheds Light on Early Evolution in Ochrophyta Plastid Organellogenesis

Phylogenetic profiling and cellular analyses of ARL16 reveal roles in traffic of IFT140 and INPP5E

Gregarine single-cell transcriptomics reveals differential mitochondrial remodeling and adaptation in apicomplexans