RESEARCH ARTICLE Open Access A multigene phylogeny of Olpidium and its implications for early fungal evolution Satoshi Sekimoto1,3*, D���Ann Rochon2, Jennifer E Long1,4, Jaclyn M Dee1 and Mary L Berbee1 Abstract Background: From a common ancestor with animals, the earliest fungi inherited flagellated zoospores for dispersal in water. Terrestrial fungi lost all flagellated stages and reproduce instead with nonmotile spores. Olpidium virulentus (= Olpidium brassicae), a unicellular fungus parasitizing vascular plant root cells, seemed anomalous. Although Olpidium produces zoospores, in previous phylogenetic studies it appeared nested among the terrestrial fungi. Its position was based mainly on ribosomal gene sequences and was not strongly supported. Our goal in this study was to use amino acid sequences from four genes to reconstruct the branching order of the early- diverging fungi with particular emphasis on the position of Olpidium. Results: We concatenated sequences from the Ef-2, RPB1, RPB2 and actin loci for maximum likelihood and Bayesian analyses. In the resulting trees, Olpidium virulentus, O. bornovanus and non-flagellated terrestrial fungi formed a strongly supported clade. Topology tests rejected monophyly of the Olpidium species with any other clades of flagellated fungi. Placing Olpidium at the base of terrestrial fungi was also rejected. Within the terrestrial fungi, Olpidium formed a monophyletic group with the taxa traditionally classified in the phylum Zygomycota. Within Zygomycota, Mucoromycotina was robustly monophyletic. Although without bootstrap support, Monoblepharidomycetes, a small class of zoosporic fungi, diverged from the basal node in Fungi. The zoosporic phylum Blastocladiomycota appeared as the sister group to the terrestrial fungi plus Olpidium. Conclusions: This study provides strong support for Olpidium as the closest living flagellated relative of the terrestrial fungi. Appearing nested among hyphal fungi, Olpidium���s unicellular thallus may have been derived from ancestral hyphae. Early in their evolution, terrestrial hyphal fungi may have reproduced with zoospores. Background Fungi in modern ecosystems are able to cause plant dis- eases, serve as mycorrhizal partners to plants, or decom- pose litter and woody debris using the tubular hyphae (filaments of walled cells) that make up fungal bodies. Hyphae use hydrostatic pressure to penetrate tough sub- strates such as soil and plant tissue, secreting enzymes across their chitinous cell walls to break down complex organic compounds into simple, diffusible molecules that are absorbed to nourish growth. An increasing body of phylogenetic evidence indicates that fungi, ani- mals, and protists, such as nucleariid amoebae and Ichthyosporea, all share a close common ancestor [1-3]. This pattern implies that the original fungus-like organisms were not terrestrial and hyphal in their assim- ilative phase but were instead aquatic, flagellated and unicellular. Fungi that have been classified in Zygomycota are phylogenetically important because in most studies, they appear as the first terrestrial fungi to have evolved from flagellated, aquatic ancestors. However, their backbone relationships remain largely unresolved. The lack of decisive evidence for monophyly has led to alternative classifications for Zygomycota [4]. Fungi once placed in Zygomycota are sometimes distributed among the Glo- meromycota, comprising the arbuscular mycorrhizal fungi [5-7] the Mucoromycotina and ���Zygomycota, unresolved���, which includes animal or fungal symbionts or pathogens in the subphyla Entomophthoromycotina, Zoopagomycotina, and Kickxellomycotina. For conveni- ence in the text, we will continue to apply ���Zygomycota��� * Correspondence: ssekimoto@bama.ua.edu 1Department of Botany, 3529-6270 University Boulevard, University of British Columbia, Vancouver, British Columbia, V6T 1Z4 Canada Full list of author information is available at the end of the article Sekimoto et al. BMC Evolutionary Biology 2011, 11:331 http://www.biomedcentral.com/1471-2148/11/331 �� 2011 Sekimoto et al licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

to these terrestrial, non-flagellated fungi including Glo- meromycota and Mucoromycotina. In terms of evolutionary inference, some of the first molecular phylogenies from ribosomal gene sequences specified, probably incorrectly, that two clades of terres- trial Zygomycota evolved convergently from flagellated, aquatic ancestors. Early studies showed that the flagel- lated Blastocladiomycota grouped with terrestrial Zygo- mycota including Rhizopus, and the flagellated Chytridiomycota grouped with the terrestrial Zygomy- cota Basidiobolus [8-11]. This pattern was likely an arti- fact of long-branch attraction and it is contradicted by more recent analyses including more taxa or different loci. More recently, a phylogeny of the amino acid sequences of RPB1 showed Basidiobolus grouping with other Zygomycota rather than with Chytridiomycota [12] Zygomycota appear monophyletic in analysis of RPB1 and RPB2 [13] and Zygomycota are paraphyletic in a multi-locus, phylogenomic study [14]. Against the background of recent support for a single origin of nonflagellated terrestrial fungi (Zygomycota plus Dikarya), James et al. [5,15] found yet another pos- sible example of convergent loss of flagella. As the first to include the zoospore-producing Olpidium virulentus (= Olpidium brassicae) [16] in their analyses, James et al. [5] were surprised to find that this flagellated fungus clustered with Basidiobolus, although without statistical support. Olpidium and Basidiobolus were further nested among terrestrial fungi with strong support from both posterior probabilities and likelihood bootstrap propor- tions. To explain the nesting of Olpidium within the non-flagellated fungi required 2-4 losses of flagella [5]. This finding of a flagellate within the terrestrial clade was no obvious artifact of long-branch attraction. The James et al. [5] study included a rich sampling of avail- able basal fungal lineages and neither Olpidium nor Basidiobolus had particularly long-branch lengths. Olpidium is however a challenging genus and it seemed possible that its apparent phylogenetic position was influenced by missing data. Several species, includ- ing Olpidium virulentus and Olpidium bornovanus, are biotrophic plant pathogens, unable to grow except as unicellular thalli that develop embedded inside living plant root cells [17-19]. At maturity, zoospores with sin- gle posterior flagella are liberated from the root cell through spore exit tubes [17] (Figure 1). Because they are biotrophic, relatively pure Olpidium DNA can only be harvested from zoospores. Washing roots with mature sporangia in distilled water triggers zoospore release. However, the zoospore suspension is not axenic. Olpidium DNA sequences are mostly too divergent to be amplified with universal fungal primers. As a result of these difficulties, James et al. [5] were only able to analyze one protein coding gene sequence (RPB1) in Figure 1 Olpidium bornovanus, a unicellular fungus, is an obligate parasite of plants that reproduces with flagellated, swimming zoospores. A-B. Vegetative unicellular thalli in cucumber root cells. Thalli differentiate into sporangia with zoospores, or into resting spores. C. An empty sporangium, after zoospore release. D. A thick-walled resting spore. E. Zoospores being released from a sporangium, showing the sporangium exit tube (arrowheads). F. A swimming zoospore with a single posterior flagellum. G. An encysted zoospore. Bars: A-E = 10 ��m F,G = 5 ��m. Sekimoto et al. BMC Evolutionary Biology 2011, 11:331 http://www.biomedcentral.com/1471-2148/11/331 Page 2 of 10