bioRxiv preprint doi: https://doi.org/10.1101/2020.08.23.262857; this version posted August 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. A genome-scale phylogeny of Fungi; insights into early evolution, radiations, and the relationship between taxonomy and phylogeny Yuanning Li1, Jacob L. Steenwyk1, Ying Chang2, Yan Wang 3,4, Timothy Y. James5, Jason E. Stajich3, Joseph W. Spatafora2, Marizeth Groenewald6, Casey W. Dunn7, Chris Todd Hittinger8, Xing-Xing Shen9,#, and Antonis Rokas1,# 1 Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee, USA 2 Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331 3 Department of Microbiology and Plant Pathology, Institute for Integrative Genome Biology, University of California, Riverside, California 92521, USA 4 Department of Biological Sciences, University of Toronto Scarborough and Department of Ecology and Evolutionary Biology, University of Toronto, Toronto, Canada 5 Department of Ecology and Evolutionary Biology, University of Michigan 48109, Ann Arbor, MI, USA 6 Westerdijk Fungal Biodiversity Institute, 3584 CT, Utrecht, The Netherlands 7 Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT 06520, USA 8 Laboratory of Genetics, Center for Genomic Science Innovation, J. F. Crow Institute for the Study of Evolution, DOE Great Lakes Bioenergy Research Center, Wisconsin Energy Institute, University of Wisconsin-Madison, Madison, WI, USA 9 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 310058, China #Address correspondence to: [email protected] & [email protected] Keywords: phylogenomics, concatenation, coalescence, polytomy test, phylogenetic signal, relative evolutionary divergence, Zygomycota, ancient diversification, taxonomy Running title: A Genome-Scale Fungal Phylogeny bioRxiv preprint doi: https://doi.org/10.1101/2020.08.23.262857; this version posted August 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Abstract Phylogenomic studies based on genome-scale amounts of data have greatly improved understanding of the tree of life. Despite their diversity, ecological significance, and biomedical and industrial importance, large-scale phylogenomic studies of Fungi are lacking. Furthermore, several evolutionary relationships among major fungal lineages remain controversial, especially those at the base of the fungal phylogeny. To begin filling these gaps and assess progress toward a genome-scale phylogeny of the entire fungal kingdom, we compiled a phylogenomic data matrix of 290 genes from the genomes of 1,644 fungal species that includes representatives from most major fungal lineages; we also compiled 11 additional data matrices by subsampling genes or taxa based on filtering criteria previously shown to improve phylogenomic inference. Analyses of these 12 data matrices using concatenation- and coalescent-based approaches yielded a robust phylogeny of the kingdom in which ~85% of internal branches were congruent across data matrices and approaches used. We found support for several relationships that have been historically contentious (e.g., for the placement of Wallemiomycotina (Basidiomycota), as sister to Agaricomycotina), as well as evidence for polytomies likely stemming from episodes of ancient diversification (e.g., at the base of Basidiomycota). By examining the relative evolutionary divergence of taxonomic groups of equivalent rank, we found that fungal taxonomy is broadly aligned with genome sequence divergence, but also identified lineages, such as the subphylum Saccharomycotina, where current taxonomic circumscription does not fully account for their high levels of evolutionary divergence. Our results provide a robust phylogenomic framework to explore the tempo and mode of fungal evolution and directions for future fungal phylogenetic and taxonomic studies. 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.23.262857; this version posted August 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Introduction Fungi comprise an estimated 2-5 million species1,2, represent one of the most diverse and ancient branches of the tree of life, and play vital roles in terrestrial and aquatic ecosystems (Fig. 1)3. Fungal organisms exhibit a wide variety of morphologies, developmental patterns, and ecologies, and are thought to have coevolved with plants through diverse modes of symbiosis, including parasitism, mutualism, and saprotrophy4. Many fungi are economically important as model organisms (e.g., the brewer’s yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe, and the bread mold Neurospora crassa); food sources (e.g., mushrooms and truffles); cell factories for the production of diverse organic acids, proteins, and natural products (e.g., the mold Aspergillus niger); or major pathogens of plants and animals (e.g., the rice blast fungus Magnaporthe grisea and the amphibian chytrid fungus Batrachochytrium dendrobatidis), including humans (e.g., Candida species causing candidiasis, Aspergillus species causing aspergillosis, and Cryptococcus species causing cryptococcosis)3,5,6. 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.23.262857; this version posted August 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. A B C D E F G H I J K L Figure 1. Diversity of major fungal lineages. Representative species for major fungal lineages. (A) Crown coral Artomyces pyxidate (Agaricomycotina, Basidiomycota). (B) Yellow brain fungus Tremella mesenterica (Pucciniomycotina, Basidiomycota). (C) Flowerpot parasol, Leucocoprinus birnbaumii (Agaricomycotina, Basidiomycota). (D) Pearl oyster mushroom, Pleurotus ostreatus (Agaricomycotina, Basidiomycota). (E) Snow fungus, Tremella fuciformis (Agaricomycotina, Basidiomycota). (F) Turkey tail, Trametes versicolor (Agaricomycotina, Basidiomycota). (G) Baker’s yeast Saccharomyces cerevisiae (Saccharomycotina, Ascomycota). (H) Fission yeast Schizosaccharomyces pombe (Taphrinomycotina, Ascomycota). (I) Mucor mucedo (Mucoromycotina, Mucoromycota). (J) Corn smut Ustilago maydis (Ustilaginomycotina, Basidiomycota). (K) Penicillium digitatum (Pezizomycotina, Ascomycota). (L) Fly agaric Amanita muscaria (Agaricomycotina, Basidiomycota). A-F, Photograph courtesy of Jacob L. Steenwyk. G, J, L, Images are available to the public domain through https://commons.wikimedia.org/wiki/. H, Photograph reproduced with permission of David O. Morgan. I, Photograph courtesy of Kerry O’Donnell and Jason Stajich. 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.23.262857; this version posted August 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. There are more than 200 orders of fungi that are classified into 12 phyla6 (for an alternative scheme of classification, see Ref.7). These 12 phyla are placed into six major groups: the subkingdoms Dikarya (which includes the phyla Ascomycota, Basidiomycota, and Entorrhizomycota) and Chytridiomyceta (which includes the phyla Chytridiomycota, Monoblepharidomycota, and Neocallimastigomycota), the phyla Mucoromycota, Zoopagomycota, and Blastocladiomycota, and the major group Opisthosporidia (which includes the phyla Aphelidiomycota, Cryptomycota/Rozellomycota, and Microsporidia, and is possibly paraphyletic)6. Evolutionary relationships among some of these groups, as well as among certain phyla and classes have been elusive, with morphological and molecular studies providing support for conflicting phylogenetic hypotheses or being equivocal in their support among alternatives6,8 (Supplementary Fig. 1). Several notable cases of phylogenetic ambiguity exist. For example, relationships among the three phyla that comprise Opisthosporidia are ambiguous, especially the placement of Aphelidiomycota (Supplementary Fig. 1). This is likely due to the parasitic lifestyles and highly reduced morphologies of many of the organisms involved that coincide with highly reduced and very rapidly evolving genomes (e.g., Microsporidia), that render their evolutionary placement challenging9,10. Ambiguity also exists with respect to the placement of Blastocladiomycota – either as a sister to the rest of fungi excluding Opisthosporidia or as a sister to the rest of fungi excluding Opisthosporidia and Chytridiomyceta6,11–13. Although aquatic, Blastocladiomycota have several traits found in terrestrial fungi, making their placement on the fungal phylogeny key for understanding the evolution of diverse