Mycologia, 108(5), 2016, pp. 1028–1046. DOI: 10.3852/16-042 # 2016 by The Mycological Society of America, Lawrence, KS 66044-8897 A phylum-level phylogenetic classification of zygomycete fungi based on genome-scale data Joseph W. Spatafora1 Jason E. Stajich Ying Chang Department of Plant Pathology & Microbiology and Institute Department of Botany and Plant Pathology, Oregon State for Integrative Genome Biology, University of California– University, Corvallis, Oregon 97331 Riverside, Riverside, California 92521 Gerald L. Benny Katy Lazarus Abstract: Zygomycete fungi were classified as a single Matthew E. Smith phylum, Zygomycota, based on sexual reproduction by Department of Plant Pathology, University of Florida, Gainesville, Florida 32611 zygospores, frequent asexual reproduction by sporangia, absence of multicellular sporocarps, and production of Mary L. Berbee coenocytic hyphae, all with some exceptions. Molecular Department of Botany, University of British Columbia, phylogenies based on one or a few genes did not support Vancouver, British Columbia, V6T 1Z4 Canada the monophyly of the phylum, however, and the phylum Gregory Bonito was subsequently abandoned. Here we present phyloge- Department of Plant, Soil, and Microbial Sciences, Michigan netic analyses of a genome-scale data set for 46 taxa, State University, East Lansing, Michigan 48824 including 25 zygomycetes and 192 proteins, and we dem- Nicolas Corradi onstrate that zygomycetes comprise two major clades Department of Biology, University of Ottawa, Ottawa, that form a paraphyletic grade. A formal phylogenetic Ontario, K1N 6N5 Canada classification is proposed herein and includes two phyla, six subphyla, four classes and 16 orders. On the basis Igor Grigoriev of these results, the phyla Mucoromycota and Zoopago- US Department of Energy (DOE) Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, California 94598 mycota are circumscribed. Zoopagomycota comprises Entomophtoromycotina, Kickxellomycotina and Zoopa- Andrii Gryganskyi gomycotina; it constitutes the earliest diverging lineage L.F. Lambert Spawn Co., Coatesville, Pennsylvania 19320 of zygomycetes and contains species that are primarily Timothy Y. James parasites and pathogens of small animals (e.g. amoeba, Department of Ecology and Evolutionary Biology, University insects, etc.) and other fungi, i.e. mycoparasites. Mucor- of Michigan, Ann Arbor, Michigan 48103 omycota comprises Glomeromycotina, Mortierellomy- Kerry O’Donnell cotina, and Mucoromycotina and is sister to Dikarya. Mycotoxin Prevention and Applied Microbiology Research It is the more derived clade of zygomycetes and mainly Unit, NCAUR-ARS-USDA, 1815 N. University Street, Peoria, consists of mycorrhizal fungi, root endophytes, and Illinois 61604 decomposers of plant material. Evolution of trophic modes, morphology, and analysis of genome-scale data Robert W. Roberson School of Life Sciences, Arizona State University, Tempe, are discussed. Arizona 85287 Key words: Entomophthoromycotina, fungi, Glo- 2 meromycotina, Kickellomycotina, Mortierellomyco- Thomas N. Taylor tina, Mucoromycota, Mucoromycotina, paraphyly, Department of Ecology and Evolutionary Biology, and Natural History Museum and Biodiversity Research Center, systematics, Zoopagomycota Zoopagomycotina University of Kansas, Lawrence, Kansas 66045 INTRODUCTION Jessie Uehling Rytas Vilgalys Despite advances in our understanding of evolutionary Biology Department, Box 90338, Duke University, Durham, relationships within Kingdom Fungi, the earliest diverg- North Carolina 27708 ing events are still poorly understood. Included among Merlin M. White these unresolved events are the evolutionary transitions Department of Biological Sciences, Boise State University, that ultimately culminated in modern diversity and in Boise, Idaho 83725 the emergence of terrestrial fungi, including subking- dom Dikarya, which comprises the phyla Ascomycota Submitted 23 Feb 2016; accepted for publication 4 Jul 2016. 1 Corresponding author. E-mail: [email protected] and Basidiomycota. Resolving the earliest branches in 2 Deceased 28 Apr 2016. the fungal genealogy is essential to identify characteristics 1028 SPATAFORA ET AL.: CLASSIFICATION OF THE ZYGOMYCETES 1029 of the ancestral fungi, to determine what traits emerged arbuscular mycorrhizal fungi, which arguably comprise with the dawn of terrestrial ecosystems, and to obtain the most successful plant-fungal symbiosis on Earth. an accurate assessment of the morphological and genetic Glomeromycota has been a phylogenetic enigma because homologies associated with fungal lifestyles. Central to itlacksanyknownformofsexualreproduction.Morpho- this transition are the fungi that were once classified logical hypotheses placed Glomeromycota among the in the phylum Zygomycota Moreau (1954). However, zygomycetes (Gerdemann and Trappe 1974, Morton because the monophyly of Zygomycota was not sup- and Benny 1990), whereas rDNA-based phylogenies ported in recent phylogenetic analyses (e.g. James et al. placed this phylum as sister to Dikarya (Schüßler et al. 2006, Liu et al. 2009, Chang et al. 2015), these fungi are 2001). Mitochondrial phylogenies (Nadimi et al. 2012, informally referred to herein as zygomycetes. Pelin et al. 2012) placed Glomeromycota as sister to Mor- Zygomycetes are filamentous, nonflagellated fungi tierellomycotina, which is supported by some but not all that mark the major transition away from the earliest genome-scale phylogenies (Tisserant et al. 2013, Chang diverging zoosporic fungi in Cryptomycota, Chytridio- et al. 2015). mycota, and Blastocladiomycota toward the rise of The second of the larger groups, “zygomycetes II”, the nonflagellated, filamentous, multicellular Dikarya. includes Entomophthoromycota, Kickxellomycotina, The zygomycetes include: (i) Phycomyces blakesleeanus and Zoopagomycotina (James et al. 2006, White et al. and other important model organisms; (ii) species 2006, Sekimoto et al. 2011, Ebersberger et al. 2012, such as Rhizopus stolonifer that cause economically Chang et al. 2015). Zygomycetes II is more difficult of significant pre- and postharvest diseases of fruits; (iii) the two groups to study. In phylogenetic analyses, it members of Glomeromycota that colonize roots and has been weakly supported (James et al. 2006, Sekimoto form endomycorrhizal symbioses with more than 80% et al. 2011) or strongly supported but based only on a of land plants; and (iv) diverse and important patho- couple of taxa (Chang et al. 2015). Entomophthoromy- gens or commensals of insects, nematodes, and other cotina, the “insect destroyers”, includes parasites of soil invertebrates (Benny et al. 2014, Redecker and insects and mites, commensals of reptiles and amphib‐ Schüßler 2014). Some zygomycetes significantly benefit ians, and poorly known parasites of desmid algae. humans by the production of compounds such as lyco- Kickxellomycotina comprises a diverse assemblage pene, fatty acids, and biodiesel, but they can also cause of fungi associated with the hindgut of arthropods, rare and deadly human diseases such as zygomycosis saprobic species with broad substrate ranges and myco- (Papanikolaou and Panayotou 2007, Wang et al. 2011, parasites. Zoopagomycotina are either obligate myco- Doggett and Wong 2014). parasites or pathogens of invertebrates, including Abandonment of the phylum Zygomycota was nematodes, rotifers, and amoebae. Members of the formalized in Hibbett et al. (2007), which treated zygomycetes II group are almost exclusively charac‐ zygomycete fungi as four subphyla incertae sedis, includ- terized by associations with animals and fungi with ing Entomophthoromycotina, Kickellomycotina, Mucor- essentially no associations with living plants, either as omycotina, and Zoopagomycotina and the phylum pathogens or symbionts (Benny et al. 2014). Glomeromycota. Mortierella was classified with the mor- Although the applications of multigene analysis phologically similar Mucorales until multigene analyses has resulted in limited phylogenetic resolution of demonstrated that it was phylogenetically distinct from zygomycetes in kingdom-level analyses, they have led Mucoromycotina, resulting in the description of the sub- to significant refinement of evolutionary hypotheses phylum Mortierollomycotina (Hoffmann et al. 2011). for selected groups of zygomycetes, based on a combi- Results from rDNA and multigene molecular phyloge- nation of molecular and morphological data. These netic studies resolved these zygomycete taxa into two include a family-level phylogenetic classification of larger groups. One of the groups, informally known as Mucorales (Hoffmann et al. 2013), testing of ordinal- “zygomycetes I”, includes Mucoromycotina and Mortier- level phylogenetic and taxonomic hypotheses for ellomycotina and in some studies, Glomeromycota Kickxellomycotina (Tretter et al. 2014) and characteri- (James et al. 2006, White et al. 2006, Chang et al. zation of the major clades of Entomophthoromycota 2015). Mucoromycotina includes Mucor, Rhizopus,and and temporal estimates of their origin in the geologic the majority of the most common and best known zygo- record (Gryganskyi et al. 2012). However, unlike mycetes. Many of these are fast growing, early colonizers Dikarya for which genome data and phylogenomic of carbon-rich substrates, with several species used in analyses have transformed our understanding of phylo- industry for organic acid production and fermen‐ genetic relationships and evolutionary processes (e.g. tation (Jennessen et al. 2008).