Molecular Phylogenetics and Evolution 65 (2012) 682–694 Contents lists available at SciVerse ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Molecular phylogeny of the Entomophthoromycota ⇑ Andrii P. Gryganskyi a,f, , Richard A. Humber b, Matthew E. Smith c, Jolanta Miadlikovska a, Steven Wu a, Kerstin Voigt d, Grit Walther d,e, Iryna M. Anishchenko f, Rytas Vilgalys a a Duke University, Department of Biology, Durham, NC 27708-90338, USA b USDA-ARS BioIPM Research, RW Holley Center for Agriculture & Health 538 Tower Road, Ithaca, NY 14853, USA c Department of Plant Pathology, University of Florida, Gainesville, FL 32611, USA d Friedrich-Schiller University of Jena, Institute of Microbiology, 25 Neugasse, Jena 07743, Germany e CBS-KNAW Fungal Biodiversity Centre, 8 Uppsalalaan, Utrecht 3584 CT, The Netherlands f M. Kholodny Institute of Botany, National Academy of Sciences of Ukraine, 2 Tereshchenkivska St., Kyiv 01601, Ukraine article info abstract Article history: The Entomophthoromycota is a ubiquitous group of fungi best known as pathogens of a wide variety of Received 28 February 2012 economically important insect pests, and other soil invertebrates. This group of fungi also includes a Revised 3 July 2012 small number of parasites of reptiles, vertebrates (including humans), macromycetes, fern gametophytes, Accepted 23 July 2012 and desmid algae, as well as some saprobic species. Here we report on recent studies to resolve the phy- Available online 2 August 2012 logenetic relationships within the Entomophthoromycota and to reliably place this group among other basal fungal lineages. Bayesian Interference (BI) and Maximum Likelihood (ML) analyses of three genes Keywords: (nuclear 18S and 28S rDNA, mitochondrial 16S, and the protein-coding RPB2) as well as non-molecular Fungi data consistently and unambiguously identify 31 taxa of Entomophthoromycota as a monophyletic group Monophyly Divergence time distinct from other Zygomycota and flagellated fungi. Using the constraints of our multi-gene dataset we Ancestral state constructed the most comprehensive rDNA phylogeny yet available for Entomophthoromycota. The taxa Substitution rate studied here belong to five distinct, well-supported lineages. The Basidiobolus clade is the earliest diverg- ing lineage, comprised of saprobe species of Basidiobolus and the undescribed snake parasite Schizangiella serpentis nom. prov. The Conidiobolus lineage is represented by a paraphyletic grade of trophically diverse species that include saprobes, insect pathogens, and facultative human pathogens. Three well supported and exclusively entomopathogenic lineages in the Entomophthoraceae center around the genera Batkoa, Entomophthora and Zoophthora, although several genera within this crown clade are resolved as non- monophyletic. Ancestral state reconstruction suggests that the ancestor of all Entomophthoromycota was morphologically similar to species of Conidiobolus. Analyses using strict, relaxed, and local molecular clock models documented highly variable DNA substitution rates among lineages of Entomophthoromy- cota. Despite the complications caused by different rates of molecular evolution among lineages, our dat- ing analysis indicates that the Entomophthoromycota originated 405 ± 90 million years ago. We suggest that entomopathogenic lineages in Entomophthoraceae probably evolved from saprobic or facultatively pathogenic ancestors during or shortly after the evolutionary radiation of the arthropods. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction mans (Pfitzer, 1872; Humber, 1981, 1984; Sharma et al., 2003; Hibbett et al., 2007; Koval, 2007). The phylum Entomophthoromycota (Humber, in press) is an eco- The Entomophthoromycota display a variety of different growth logically important fungal lineage consisting of more than 180 spe- forms. These fungi can grow as a well-developed, multinucleate or cies that are primarily obligate arthropod parasites that are found septate mycelium that is either walled or protoplastic, depending in diverse habitats worldwide. This group also includes some sap- on the species and the stage of growth. Most of the entomopatho- rotrophic taxa as well as a handful of species that are parasites of genic species develop inside their arthropod hosts as multinucleate desmid algae, fern gametophytes, tardigrades, and nematodes as hyphal bodies. Asexual reproduction in species of Entomophthor- well as several facultative parasites of vertebrates including hu- omycota is characterized by the production of forcibly discharged conidia on distinctive conidiophores, and the routine formation of actively or passively dispersed secondary conidia. This repetitive production of forcibly discharged conidia is unique among basal ⇑ Corresponding author at: Duke University, Department of Biology, Durham, NC 27708-90338, USA. fungal lineages and appears to be a synapomorphy. The thick- E-mail address: [email protected] (A.P. Gryganskyi). walled resting spores form either as zygospores after the fusion 1055-7903/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ympev.2012.07.026 A.P. Gryganskyi et al. / Molecular Phylogenetics and Evolution 65 (2012) 682–694 683 of compatible hyphae (gametangia) or, most commonly in Entom- ophthoromycota and across all early diverging fungi (Nagahama ophthoraceae, as azygospores formed without a prior gametangial et al., 1995; Jensen et al., 1998). Traditional taxonomy had recog- conjugation. It is difficult to determine whether any of these rest- nized the large, diverse, and heterogeneous phylum Zygomycota ing spores is the result of sexual reproduction since it appears that for fungi with non-septate mycelium and sexual spores formed the morphological events of conjugation and genetic events of after hyphal fusion (Moreau, 1954). Molecular phylogenetic stud- karyogamy and meiosis may not be obligatorily linked in these ies have verified the heterogeneity of this phylum by recognizing fungi (Humber, 1981, in press; McCabe et al., 1984). It has been at least five apparently monophyletic groups within the fungi of suggested that all Entomophthoromycota species are homothallic Zygomycota s.l. Based on the results of a six-gene phylogenetic but the mating biology of this group has not been studied geneti- analysis of all major fungal groups (James et al., 2006), the phylum cally (Humber, in press). Intracellular characteristics such as nucle- Zygomycota was rejected as non-monophyletic and these five ar size and number, size and position of the nucleolus, and the molecularly distinct lineages were recognized as the phylum Glom- details of mitotic divisions have long been used to differentiate eromycota (arbuscular mycorrhizal fungi) and four subphyla of the taxa, particularly at the familial rank (Humber, 1981, 1984, uncertain position (incertae sedis): Mucoromycotina, Entomopht- 1989). horomycotina, Kickxellomycotina, and Zoopagomycotina (Hibbett The molecular systematics of the Entomophthoromycota was et al., 2007). Fifth subphyllum Mortierellomycotina was separated first addressed in the 1990s when a few taxa were included in from Mucoromycotina recently (Hoffmann et al., 2011). Nomencla- analyses to elucidate broad phylogenetic patterns in the evolution ture for the major groups of former Zygomycota has not yet been of basal fungi. The early phylogenetic studies repeatedly revealed a standardized (e.g., Kirk et al., 2001; O’Donnell et al., 2001; Hibbett striking lack of concordance between traditional morphological et al., 2007). While evolutionary relationships within the Entom- taxonomy and the newer molecular data, both within the Entom- ophthoromycotina have not been fully resolved, the morphology Fig. 1. Placement of Entomophthoromycota among the basal fungal lineages in 12 previously published studies. Thickened branches indicate lineages with statistically significant support as reported in the original publication. Filled ovals indicate Entomophthoromycota and small open circles with tails indicate flagellated fungi (Chytridiomycota and Blastocladiomycota). Phylogenies are modified for presentation here based on the following publications: 1 – Nagahama et al. (1995);2–Jensen et al. (1998);3–James et al. (2000);4–Tanabe et al. (2000);5–Keeling (2003);6–Einax and Voigt (2003);7–Tanabe et al. (2004);8–James et al. (2006);9–White et al. (2006); 10 – Liu and Voigt (2010);11–Ebersberger et al. (2011);12–Sekimoto et al. (2011). 684 A.P. Gryganskyi et al. / Molecular Phylogenetics and Evolution 65 (2012) 682–694 of this group and the results of James et al. (2006) suggst that it gle gene suggested a polyphyletic Entomophthoromycota, with may be a unique monophyletic lineage. Based on these criteria, Basidiobolus split from the rest of the group (Nagahama et al., Humber (in press) reasoned that this lineage was sufficiently dis- 1995; Jensen et al., 1998)(Fig. 1.1–7 and 1.10). In contrast, mul- tinct to be recognized at the phylum rank as Entomophthoromycota. ti-locus phylogenetic analyses have delimited a monophyletic Previous molecular studies suggest that the Entomophthoromy- Entomophthoromycota (Fig. 1.8: James et al., 2006; Fig. 1.9: White cota is a polyphyletic group with uncertain placement of its main et al., 2006). Another characteristic feature of most phylogenetic lineages (Nagahama et al., 1995; Jensen et al., 1998; James et al., studies is that most have reconstructed flagellated fungi as a ‘‘sister 2006; White et al., 2006; Liu