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A multigene phylogeny of Olpidium and its implications for early fungal evolution Sekimoto, Satoshi; Rochon, D’Ann; Long, Jennifer E; Dee, Jaclyn M; Berbee, Mary L Nov 15, 2011

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RESEARCH ARTICLE Open AccessA multigene phylogeny of Olpidium and itsimplications for early fungal evolutionSatoshi Sekimoto1,3*, D’Ann Rochon2, Jennifer E Long1,4, Jaclyn M Dee1 and Mary L Berbee1AbstractBackground: From a common ancestor with animals, the earliest fungi inherited flagellated zoospores for dispersalin water. Terrestrial fungi lost all flagellated stages and reproduce instead with nonmotile spores. Olpidiumvirulentus (= 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 terrestrialfungi. Its position was based mainly on ribosomal gene sequences and was not strongly supported. Our goal inthis 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 Bayesiananalyses. In the resulting trees, Olpidium virulentus, O. bornovanus and non-flagellated terrestrial fungi formed astrongly supported clade. Topology tests rejected monophyly of the Olpidium species with any other clades offlagellated 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. WithinZygomycota, Mucoromycotina was robustly monophyletic. Although without bootstrap support,Monoblepharidomycetes, a small class of zoosporic fungi, diverged from the basal node in Fungi. The zoosporicphylum 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 theterrestrial fungi. Appearing nested among hyphal fungi, Olpidium’s unicellular thallus may have been derived fromancestral hyphae. Early in their evolution, terrestrial hyphal fungi may have reproduced with zoospores.BackgroundFungi 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 enzymesacross their chitinous cell walls to break down complexorganic compounds into simple, diffusible moleculesthat are absorbed to nourish growth. An increasingbody of phylogenetic evidence indicates that fungi, ani-mals, and protists, such as nucleariid amoebae andIchthyosporea, all share a close common ancestor [1-3].This pattern implies that the original fungus-likeorganisms were not terrestrial and hyphal in their assim-ilative phase but were instead aquatic, flagellated andunicellular.Fungi that have been classified in Zygomycota arephylogenetically important because in most studies, theyappear as the first terrestrial fungi to have evolved fromflagellated, aquatic ancestors. However, their backbonerelationships remain largely unresolved. The lack ofdecisive evidence for monophyly has led to alternativeclassifications for Zygomycota [4]. Fungi once placed inZygomycota are sometimes distributed among the Glo-meromycota, comprising the arbuscular mycorrhizalfungi [5-7]; the Mucoromycotina; and “Zygomycota,unresolved”, which includes animal or fungal symbiontsor pathogens in the subphyla Entomophthoromycotina,Zoopagomycotina, and Kickxellomycotina. For conveni-ence in the text, we will continue to apply ‘Zygomycota’* Correspondence: ssekimoto@bama.ua.edu1Department of Botany, 3529-6270 University Boulevard, University of BritishColumbia, Vancouver, British Columbia, V6T 1Z4 CanadaFull list of author information is available at the end of the articleSekimoto et al. BMC Evolutionary Biology 2011, 11:331http://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 CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction 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 firstmolecular phylogenies from ribosomal gene sequencesspecified, 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 flagellatedChytridiomycota grouped with the terrestrial Zygomy-cota Basidiobolus [8-11]. This pattern was likely an arti-fact of long-branch attraction and it is contradicted bymore recent analyses including more taxa or differentloci. More recently, a phylogeny of the amino acidsequences of RPB1 showed Basidiobolus grouping withother Zygomycota rather than with Chytridiomycota[12]; Zygomycota appear monophyletic in analysis ofRPB1 and RPB2 [13]; and Zygomycota are paraphyleticin a multi-locus, phylogenomic study [14].Against the background of recent support for a singleorigin of nonflagellated terrestrial fungi (Zygomycotaplus Dikarya), James et al. [5,15] found yet another pos-sible example of convergent loss of flagella. As the firstto include the zoospore-producing Olpidium virulentus(= Olpidium brassicae) [16] in their analyses, James etal. [5] were surprised to find that this flagellated fungusclustered with Basidiobolus, although without statisticalsupport. Olpidium and Basidiobolus were further nestedamong terrestrial fungi with strong support from bothposterior probabilities and likelihood bootstrap propor-tions. To explain the nesting of Olpidium within thenon-flagellated fungi required 2-4 losses of flagella [5].This finding of a flagellate within the terrestrial cladewas no obvious artifact of long-branch attraction. TheJames et al. [5] study included a rich sampling of avail-able basal fungal lineages and neither Olpidium norBasidiobolus had particularly long-branch lengths.Olpidium is however a challenging genus and itseemed possible that its apparent phylogenetic positionwas influenced by missing data. Several species, includ-ing Olpidium virulentus and Olpidium bornovanus, arebiotrophic plant pathogens, unable to grow except asunicellular thalli that develop embedded inside livingplant root cells [17-19]. At maturity, zoospores with sin-gle posterior flagella are liberated from the root cellthrough spore exit tubes [17] (Figure 1). Because theyare biotrophic, relatively pure Olpidium DNA can onlybe harvested from zoospores. Washing roots withmature sporangia in distilled water triggers zoosporerelease. However, the zoospore suspension is not axenic.Olpidium DNA sequences are mostly too divergent tobe amplified with universal fungal primers. As a resultof these difficulties, James et al. [5] were only able toanalyze one protein coding gene sequence (RPB1) inFigure 1 Olpidium bornovanus, a unicellular fungus, is an obligate parasite of plants that reproduces with flagellated, swimmingzoospores. 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 thesporangium exit tube (arrowheads). F. A swimming zoospore with a single posterior flagellum. G. An encysted zoospore. Bars: A-E = 10 μm; F,G= 5 μm.Sekimoto et al. BMC Evolutionary Biology 2011, 11:331http://www.biomedcentral.com/1471-2148/11/331Page 2 of 10addition to ribosomal genes, from only one Olpidiumspecies.Our objectives in this study included rigorous testingof the phylogenetic position of Olpidium and resolvingthe relationships among clades in the Zygomycota andthe flagellated fungi, with the overall goal of improvingunderstanding of the early evolution of Fungi. We usedgenes for four proteins, translation elongation factor 2(Ef-2), RNA polymerase II largest subunit (RPB1), RNApolymerase II second largest subunit (RPB2), and actin.Although Ef-2 genes have proven useful in other eukar-yotic lineages [20], this study represents their first usefor the deep phylogeny of Fungi. Olpidium, if nestedwithin Zygomycota, becomes a key organism for recon-structing the trail of how terrestrial fungi lost their fla-gella and colonized land.ResultsOverall phylogenetic analyses of the kingdom FungiThe kingdom Fungi formed a robust clade in maximumlikelihood and Bayesian analysis from the dataset of con-catenated amino acid sequences from four genes (Figure2). The terrestrial fungi plus the flagellated fungi Olpi-dium virulentus and O. bornovanus formed a monophy-letic group excluding all other flagellated fungi with 95%bootstrap support and a posterior probability of 1.0 (Fig-ure 2). Topology tests rejected all alternative trees thatconstrained the Olpidium species to cluster with othergroups of flagellated fungi (Table 1). A clade includingthe Zygomycota plus Olpidium was also monophyleticwith 68% bootstrap support from likelihood and a pos-terior probability of 0.98 (Figure 2). In our analysis, ter-restrial fungi were divided between the monophyleticZygomycota plus Olpidium, and the Dikarya (= Asco-mycota plus Basidiomycota). The Zygomycota includedtwo well-supported groups: Mucoromycotina and Glo-meromycota. The Zygomycota also included “Zygomy-cota, unresolved” (Figure 2), a weakly supported cladeconsisting largely of animal or fungal symbionts orpathogens [4-6]. “Zygomycota, unresolved” also includedOlpidium. If any group within Zygomycota were the sis-ter group to Olpidium, it was not clear from our phylo-genies. The two species that clustered most closely withOlpidium in the Bayesian analysis, Piptocephalis corym-bifera and Rhopalomyces elegans, had long-branches(data not shown), were missing data from two loci, anddid not cluster with Olpidium in the likelihood analysis(Figure 2). In Approximately Unbiased tree topologytests, uniting Olpidium with Glomeromycota could notbe rejected at the p-value of < 0.05 (Table 1). In themore conservative weighted Shimodaira-Hasegawa tests,uniting Olpidium with Mucoromycotina and withDikarya could not be rejected either (Table 1). Theseanalyses suggest that Olpidium is part of the terrestrialfungi, but leave open the possibility that it may be thesister taxon to the Dikarya, or sister to one of the basalclades within Zygomycota.Concerning the other flagellated fungi, the concate-nated dataset provided strong support for the mono-phyly of the Blastocladiomycota, the “core chytridclade”, and the Monoblepharidomycetes. Althoughsupport was low, the flagellated fungi form a paraphy-letic assemblage, with Monoblepharidomycetes diver-ging first, then the Neocallimastigomycota, the “corechytrid clade,” and finally, the Blastocladiomycota asthe sister group to the terrestrial fungi. Tree topologytests did not reject any of the alternative possible pat-terns of relationships among the three basal clades offlagellated fungi (Monoblepharidomycetes, Neocalli-mastigomycota, and “core chytrid clade”) (data notshown).Analyzed separately, individual gene trees did notresolve branching order of basal fungal clades with sig-nificant bootstrap support (Figure S1a-S1d in Additionalfile 1). However, the Ef-2 likelihood tree showed Olpi-dium in “Zygomycota, unresolved” with 56% supportand all the genes except actin placed Olpidium withmembers of “Zygomycota, unresolved”. Overall, actinprovided less phylogenetic information than the othergenes due to its low substitution rates (note the muchlonger scale bar relative to 0.1 substitutions, Figure S1d)compared with scale bars for other single genes (FigureS1a-S1c) for phylogenetic analysis.Olpidium sequencesBecause Olpidium cultures were not axenic, we had touse phylogenetic concordance as a criterion for testingwhether or not their DNA sequences could be fromcontaminants. Reassuringly, in a maximum likelihoodtree from concatenated data (Figure 2), sequences of thetwo Olpidium species formed a robust clade amongother fungi with 100% bootstrap support. As expected,they grouped among the Zygomycota, consistent withresults from James et al. [5]. Sequences from both spe-cies also clustered together in individual gene treeswhenever both species were included in the analysis(Figure S1a, S1b, and S1d in Additional file 1). TheRPB1 gene sequence obtained from our O.virulentus(GenBank AB644405) was almost identical to O.virulen-tus isolate used in James et al. (GenBank DQ294609)[5]. As an additional assay for possible contamination,we amplified the SSU rRNA gene region from DNAextracts by PCR with eukaryotic-universal SSU primers[SR1 (5’-TACCTGGTTGATCCTGCCAG-3’) and SR12(5’-CCTTCCGCAGGTTCACCTAC-3’)], then sequencedamplicons directly. We found the Olpidium SSU rRNAgene region in our extracts and we did not detect thehost plant or any other sequences (data not shown).Sekimoto et al. BMC Evolutionary Biology 2011, 11:331http://www.biomedcentral.com/1471-2148/11/331Page 3 of 10Puccinia graminisCoprinopsis cinereaPhanerochaete chrysosporiumCryptococcus neoformansNeurospora crassaYarrowia lipolyticaSaccharomyces cerevisiaeCandida albicansMortierella verticillataEndogone pisiformisUmbelopsis ramannianaPhycomyces blakesleeanusCokeromyces recurvatusRhizopus oryzaeDimargaris bacillisporaCoemansia reversaSpiromyces aspiralisFurculomyces boomerangusSmittium culisetaeConidiobolus coronatusEntomophthora muscaeBasidiobolus ranarumBasidiobolus haptosporusPiptocephalis corymbiferaRhopalomyces elegansOlpidium bornovanusOlpidium virulentus Scutellospora heterogamaGlomus intraradicesPhysoderma maydisCoelomomyces stegomyiaeBlastocladiella emersoniiCatenaria anguillulaeAllomyces arbusculusAllomyces macrogynusNeocallimastix frontalisPhlyctochytrium  planicorneChytriomyces hyalinusRhizoclosmatium sp.Rhizophlyctis roseaPolychytrium aggregatumEntophlyctis confervae-glomerataeGaertneriomyces semiglobiferusSpizellomyces punctatusRhizophydium macroporosumBatrachochytrium dendrobatidisSynchytrium macrosporumCladochytrium replicatumNowakowskiella elegansHyaloraphidium curvatumMonoblepharis macrandraMonoblepharis polymorphaGonapodya sp.Caenorhabditis elegansDrosophila melanogasterHomo sapiensDictyostelium discoideumToxoplasma gondiiCryptosporidium parvumPhytophthora sojaeCyanidioschyzon merolaePopulus trichocarpa0.11.00/1001.00/1001.00/1001.00/95-/101.00/1001.00/1001.00/1001.00/1001.00/991.00/991.00/1001.00/1001.00/921.00/1001.00/1001.00/791.00/831.00/941.00/861.00/1000.92/34“Zygomycota, unresolved”plus OlpidiumMucoromycotinaGlomeromycotaBlastocladiomycota“Core chytrid clade”NeocallimastigomycotaMonoblepharido-mycetesOther eukaryotes (Outgroup)Animalia1.00/871.00/1001.00/1001.00/93-/180.98/71-/201.00/1000.98/781.00/1000.98/681.00/871.00/1001.00/1001.00/1001.00/971.00/1000.99/341.00/971.00/1001.00/1000.99/691.00/931.00/1001.00/1001.00/1001.00/100 -/320.75/340.96/93-/26-/22-/231.00/93-/560.90/20-/36Asco-mycotaTerrestrial (non-flagellated) fungi + OlpidiumAquatic (flagellated) fungi Basidio-mycotaDikarya“Chytrids”Zygomycota•  cell with a posterior    flagelllum (         )•  hyphae (       ) •  yeast (          )Legend:Figure 2 A phylogeny from four protein-coding genes shows that Olpidium is the only flagellated genus in a clade of terrestrial non-flagellated fungi. This maximum likelihood tree from RAxML is based on concatenated amino acid sequences of genes for the elongationfactor 2, the RNA polymerase II largest and second largest subunits, and actin. Numbers on the internal nodes represent posterior probabilitiesand bootstrap percentages calculated by MrBayes and RAxML, respectively.Sekimoto et al. BMC Evolutionary Biology 2011, 11:331http://www.biomedcentral.com/1471-2148/11/331Page 4 of 10This result suggested that Olpidium DNA predominatedin our extracts.However, along with the Olpidium sequences thatclustered, as expected, in the fungi, we also found addi-tional, aberrant sequences for Ef-2, RPB2, and actin(Figure S1a, S1c, and S1d in Additional file 1). Thesesequences did not match common laboratory contami-nants. From the Ef-2 dataset, in addition to the set ofOlpidium sequences that clustered as expected, within“Zygomycota, unresolved,” a pair of sequences clusteredwith the slime mold Dictyostelium discoideum (FigureS1a). From the RPB2 dataset, one O. bornovanussequence clustered with the zygomycete Conidioboluscoronatus and the other with D. discoideum (FigureS1c). In the actin dataset, we detected two O. virulentusand four O. bornovanus actin-like sequences (FigureS1d). One pair of sequences from Olpidium speciesclustered together in Fungi, but the other foursequences clustered with non-fungal taxa (Figure S1d).The aberrant sequences could be divergent paralogs orgenes gained through horizontal gene transfer, and wecannot even rule out contamination as their source. Wedeleted those aberrant sequences (Figure S1a, S1c, S1d)from our four-protein dataset used in the likelihood andBayesian analyses (Figure 2, S2).DiscussionPhylogenetic position of OlpidiumOur likelihood and Bayesian analyses strongly suggestthat Olpidium virulentus and O. bornovanus are moreclosely related to terrestrial fungi than to other clades offlagellated fungi. Olpidium has typical “core chytrid”characters including a single endobiotic sporangiumproducing zoospores having a single posterior flagellum.It shares no obvious morphological characters with anyclade of terrestrial fungi. Whether Olpidium and terres-trial fungi share biochemical characters, such as enzymesystems for entry into plant cell walls, remains to beseen.None of the other Zygomycota genera are close toOlpidium, based on the tendency of Olpidium to clusterwith different genera in different analyses, without sup-port. This lack of resolution may be yet another long-branch attraction problem. Adding more species ofOlpidium to a phylogeny would contribute to breakingup the Olpidium branch and would allow reconstructionof the ancestral traits of Olpidium. However, most ofthe ~50 Olpidium species are difficult to obtain. Wewere able to include O. virulentus and O. bornovanus inthis study only because Rochon and colleagues growthem routinely to serve as vectors for experimentaltransmission of viral diseases of plants [21,22]. Otherspecies of evolutionary interest from other hosts (fungi,moss protonemata, microscopic animals, or algae)would have to be isolated from nature and culturedalong with their hosts. As shown in other morphologi-cally simple but diverse fungal genera, e.g., Rhizophy-dium [23], Olpidium is likely not monophyletic. Speciesdiffer in cell size (depending on the size of the hostcell), shape (spherical to ovoid, irregular, tubular orelongate), and other morphological characters (restingspore morphology, number of spore exit tubes etc.).Ultrastructure also varies from species to species. Olpi-dium pendulum perhaps belongs in a genus separatefrom O. virulentus and O. bornovanus (as a synonym ofO. cucurbitacearum) [24-31]. Some of the other speciesmight belong in the same clade as our two plant parasi-tic Olpidium, while others will likely fall out with the“core chytrids” instead.Terrestrial fungal relationships–a classical phylogeneticchallenge?Conflicts among phylogenies from different datasetssuggest that as fungi first colonized land, at least fiveTable 1 The only phylogenetic positions of Olpidium that could not be rejected by either the weighted Shimodaira-Hasegawa (wSH) or the Approximately Unbiased (AU) tests were within the Zygomycota.Tree Constraint ΔlnL AU1 wSH11 Olpidium united with “Zygomycota, unresolved” (Best tree; Figure 2) 0.0 0.896 0.9952 Olpidium united with Glomeromycota in Zygomycota 19.8 0.208 0.6193 Olpidium united with Mucoromycotina in Zygomycota 42.3 0.013* 0.0874 Olpidium united with Dikarya 65.7 0.011* 0.0585 Olpidium sister to all terrestrial fungi 80.8 0.000** 0.010*6 Olpidium united with Blastocladiomycota 148.4 0.002** 0.004**7 Olpidium sister to all other fungi 148.7 0.005** 0.008**8 Olpidium sister to all terrestrial fungi and Blastocladiomycota 154.6 0.000** 0.000**9 Olpidium united with Neocallimastigomycota 174.1 0.004** 0.013*10 Olpidium united with Monoblepharidomycetes 203.6 0.000** 0.000**11 Olpidium united with “Core chytrid clade” 233.6 0.000** 0.000**1AU, Approximately Unbiased; wSH, weighted Shimodaira-Hasegawa test. The constrained tree was significantly worse than the best tree (Figure 2) at P < 0.05*or P < 0.01**.Sekimoto et al. BMC Evolutionary Biology 2011, 11:331http://www.biomedcentral.com/1471-2148/11/331Page 5 of 10basal lineages (Mucoromycotina, Glomeromycota,“Zygomycota, unresolved”, Dikarya, Olpidium) radiatedrapidly and then evolved independently by differentrates and modes of substitution. A succession ofresearchers have noted and attempted to solve the pro-blem of variation in rates and modes with various analy-tical strategies. Tanabe et al. [32] pointed out theribosomal substitution rates varied dramatically in rela-tive rate tests and recommended relying more heavilyon RPB1, which showed less rate variation. Voigt andWöstemeyer [33] used logdet methods to overcomebiases resulting from lineage-specific variation innucleotide composition to estimate distances. Liu et al.[14] applied huge amounts of sequence, analyzing40,925 amino acids with a substitution model intendedto minimize long-branch attraction problems. In theirribosomal gene phylogeny, White et al. [6] included anexcellent sampling of taxa representing most knownZygomycota lineages, including many species that havenot appeared in other studies because they are difficultto grow.No aspect of basal branching order for these taxareceives consistent support across studies. The cladecorresponding to the traditional Zygomycota from ourstudy and from Liu et al.’s RPB1 and RPB2 amino acidphylogenies [13] does not appear in analyses consistinglargely or entirely of ribosomal DNA sequences [5,6,15]or of actin plus Ef-1 alpha [33,34]. It is also missingfrom a phylogenomic study of nuclear genes [14] whereMucoromycotina is sister to Dikarya. Our analyses showMortierella verticillata as part of Mucoromycotina. Thisrelationship was supported strongly in our analysis ofconcatenated data; it was evident in our Ef-2 and RPB1gene trees, and it is consistent with some traditionalclassifications schemes based on morphology [35]. Liu etal. [14], however, showed Mortierella diverging muchearlier (although with limited support) as one of thethree Zygomycota lineages paraphyletic to Mucoromy-cotina plus Dikarya. Although the Liu et al. [14] studyhad a great deal of data per taxon and an appropriatemodel of evolution, the number of genomes availablefor analysis may still be too small to capture branchingorder with statistical support. As a result, in Liu et al.’s[14] study, long-branch attraction or other kinds of sys-tematic error may have been responsible for pullingMortierella away from its closest relatives. In contrast,our phylogenies have good representation of lineagesbut data from only four loci, and our inability to rejectalternative topologies in weighted Shimodaira-Hasegawaand Approximately Unbiased tests reflects the need formore data per taxon. Resolution of the relationshipsamong early fungi joins the resolution of relationshipsamong the first animals and among seed plants as a dif-ficult phylogenetic problem.Phylogenetic age of origin of fungal hyphaeBranching order among early-diverging fungal groupshas been assumed to involve progressive elaboration ofthread-like hyphal systems [2], from ancestors, whichlike most “core chytrids,” had unicellular thalli. Beyondthe Fungi, the oomycetes, chromistan fungus-like pro-tists, provide an example of convergent origin of hyphae.Oomycete thalli range from single cells to well-definedmycelia [19]. Recent phylogenetic studies have indicatedthat the ancestral oomycetes might have been unicellu-lar endoparasites of marine organisms that later gaverise to hyphal species. Of the hyphal oomycete species,some remained aquatic, while others invaded land [36].Ability to form hyphae may have been an importantcharacter that allowed early oomycetes as well as earlyFungi to poke, penetrate and explore to find terrestrialfood that may have been patchy in its distribution.As in the oomycetes, hyphae may have evolved infungi even before they colonized land. Most of theknown Monoblepharidomycetes are hyphal, and theydiverged at or near the base of the Fungi. The Blastocla-diomycota, the aquatic sister clade to the terrestrialfungi [5,6,14], also includes genera with well-developedmycelia. Since its relatives are hyphal, Olpidium’s unicel-lular thallus is perhaps an adaptation to parasitism and areduction from a hyphal ancestor. On the other hand,finding Olpidium nested among the terrestrial cladesimplies that fungi on land initially had flagellated spores.Eukaryotic flagella are complex structures that wouldhave been unlikely to evolve repeatedly, and so multiplelosses of flagella were far more likely than a convergentgain in Olpidium. As in early terrestrial animals andplants, early terrestrial fungi retained a motile unicellu-lar phase.We reluctantly excluded two interesting basal fungallineages, microsporidia and Rozella [5], from our ana-lyses. Microsporidia, obligate intracellular pathogens ofanimals, have unusual, highly divergent genes [37]. Con-served synteny among the microsporidia and the zygo-mycotan Phycomyces blakesleeanus and Rhizopus oryzaehas been used to suggest that the microsporidia may berelated to Mucoromycotina [38], an interpretation con-tested by Koestler and Ebersberger [39]. Sequences frommicrosporidia are difficult to align and a source of arti-facts involving long-branch attraction. It seemed morelikely that microsporidia would obscure phylogeneticsignal in our dataset than that our analysis would cor-rectly resolve their relationships.Rozella allomycis is a unicellular obligate endoparasiteof Allomyces arbusculus, another fungus [40]. James etal. [5] found Rozella allomycis to group with microspori-dia at the base of Fungi. Unfortunately, the only knownliving culture of Rozella had died before our study, sowe were unable to contribute new data for the genus.Sekimoto et al. BMC Evolutionary Biology 2011, 11:331http://www.biomedcentral.com/1471-2148/11/331Page 6 of 10Rozella is basal in Fungi in our RPB1 and RPB2 indivi-dual trees. This could be its correct phylogenetic posi-tion but it could also reflect long-branch attraction(Figure S1b, S1c, in Additional file 1). Adding Rozella tothe analysis caused relationships among the other flagel-lated fungi to shift (Figure S2 in Additional file 2). Ourtree topology test did not rule out most possible topolo-gies for the positioning of Rozella with other fungallineages (Table S1, in Additional file 2). Uncertain aboutthe reason for the behaviour of Rozella, we show theconsequences of including it in the supplemental file(Figure S2) but excluded the taxon from the analyses inFigure 2.ConclusionsEven with analysis of amino acid sequences from fourprotein-coding loci, and from all available early-diver-ging lineages, the resolution of branching order in thedeepest parts of kingdom Fungi remains uncertain.Instead of converging on a single solution, recent studiesof early fungi have produced conflicting phylogenies.Our trees showed the zoosporic Monoblepharidomy-cetes to be sister to all other fungi, the zoosporic Blasto-cladiomycota to be sister to terrestrial fungi, and theZygomycota to be monophyletic. These relationshipsrequire further testing through phylogenomic analysisand comprehensive taxon sampling.Our study provided the strongest support to date forthe monophyletic group that includes two zoosporicOlpidium species together with all of the non-flagellated,terrestrial fungi. Here, the results from our study andothers are congruent. If its nested position amonghyphal fungi is correct, the unicellular thallus of Olpi-dium may have evolved by reduction from hyphae, con-tradicting an intuitive expectation that the smallerstructure would predate the larger one. Swimming zoos-pores that germinated and grew as hyphae may havebeen the fungal analogues of amphibians that made theearliest evolutionary forays into drier environments.MethodsFungal strains, DNA and RNA extraction, PCRamplification and sequencingTable S2 (in Additional file 3) lists fungal strainssequenced in this study. All zygomycetes were main-tained on potato dextrose agar medium (PDA: DifcoGrade, Becton, Dickinson and Company, MD, USA), orPDA plus 0.5% yeast extract medium at ambient tem-perature, as described in O’Donnell [41]. All chytridsexcept the two Olpidium strains were maintained onPmTG agar medium [42]: 1g peptonized milk, 1g tryp-tone, 5g glucose, 8g agar, and 1litre of distilled water) atambient temperature. Olpidium bornovanus was main-tained on cucumber roots (C. sativis cv. Poinsette 76),and O. virulentus was maintained on lettuce roots (Lac-tuca sativa cv. White Boston) as described by Campbellet al. [43]. The full length of the internal transcribedspacer (ITS) regions (between the 18S, 5.8S and 28Sribosomal RNA genes) of our O. virulentus strain wassequenced for species identification, and it had 100%nucleotide similarity with that of O. virulentus strainGBR1 (GenBank no. AY373011) [16,44]. Ef-2 genesequences of two Olpidium species and RPB2 genesequences of O. bornovanus were obtained from totalRNA, and all other sequences obtained in this studywere from total genomic DNA. For the RNA and DNAextraction from two Olpidium strains, we used TRIzolReagent following the procedure outlined by Invitrogen(Mississauga, Ontario, Canada). Prior to DNA and RNAextraction the Olpidium zoospores were pelleted fromroot washings at 2,700 × g for 7 minutes. Total genomicDNA from cultured strains was extracted using aDNeasy Plant Mini Kit (Qiagen, Mississauga, Ontario,Canada), following the manufacturer’s protocol.Primers were designed in this study, or taken fromJames et al. [5], or Hoffmann et al. [45] (Table S2, Addi-tional file 3 Table S3, Additional file 4). We amplifiedthe partial genes for eukaryotic translation elongationfactor 2 (Ef-2), RNA polymerase II largest subunit(RPB1), RNA polymerase II second largest subunit(RPB2), and actin using 0.5 μM concentrations of pri-mers, two to five μl of genomic DNA solution, andPureTaq™ Ready-To-Go™ PCR beads (AmershamBiosciences, Piscataway, NJ, USA) following the manu-facturer’s protocol. Total PCR reaction volume was 25μl, and cycling parameters were: initial denaturation (5min, 94°C), followed by 40 cycles (94°C, 10 s; 50-65°C,20 s; 72°C for 30 s plus 4 additional seconds per cycle),and then a final extension at 72°C for 7 min. RT-PCRwas conducted using SuperScript™ One-Step RT-PCRwith Platinum® Taq (Invitrogen) following manufac-turer’s protocol. The total 25 μl reaction volume of RT-PCR, contained 0.2 μM concentrations of primers, 1 μlof total RNA solution, 0.5 μl of RNaseOUT® Recombi-nant Ribonuclease Inhibitor (Invitrogen), 12.5 μl of 2×Reaction Mix, and 1.0 μl of RT/Platinum® Taq Mix.Cycling parameters were: reverse transcription (32 min,55°C) and initial denaturation (5 min, 94°C), followed by40 cycles (94°C, 15 s; 55°C, 30 s; 68°C for 30 s plus 4additional seconds per cycle), and then a final extensionat 68°C for 10 min. PCR products were then purifiedwith EtOH precipitation (20 μl PCR products, 2 μl 3 Msodium acetate (pH 4.5), 50 μl 95% ethanol; 15 min spinand rinse with 70% ethanol twice, resuspended in 20 μlwater), or cloned with TOPO® TA Cloning kit (Invitro-gen) following the manufacturer’s protocol. Productswere then sequenced with BigDye Terminator v3.1Cycle Sequencing Kit on a Applied Biosystems 3730SSekimoto et al. BMC Evolutionary Biology 2011, 11:331http://www.biomedcentral.com/1471-2148/11/331Page 7 of 1048-capillary sequencer (Applied Biosystems, Foster City,CA, USA) at NAPS Unit, MSL, University of BritishColumbia [accession numbers; DDBJ:AB609150 -AB609186, AB625456, and GenBank:HM117701 -HM117719; Table S4 in Additional file 5].Sequence alignmentsDNA sequences were assembled and edited using thesoftware Se-Al v2.0a11 [46]. Also using Se-Al, sequenceswere manually added to RPB1 and RPB2 amino acidalignments of James et al. [5] or to the actin alignmentof Voigt & Wöstemeyer [33]. Introns, ambiguouslyaligned positions and gaps were excluded from bothanalyses. Alignments have been accessioned in Tree-BASE (S11208, http://purl.org/phylo/treebase/phylows/study/TB2:S11208).Molecular phylogenetic analysisPhylogenetic relationships were inferred from maximumlikelihood and Bayesian methods, and all four proteindatasets were combined and used for both analyses (Fig-ure 2). ProtTest version 2.4 [47] estimated that the best-fit model of protein evolution for each of the individualalignment datasets was the LG model, with site-to-siterate variation approximated with a gamma distribution(G) and an estimated proportion of invariable sites (I),and with empirical base frequencies (F) (LG+G+I+F).For likelihood, we used RAxML version 7.0.3 [48], withthe LG+G+I+F model and 600 replicate searches. Weused 1000 likelihood bootstrap replicates with the rapidbootstrapping algorithm in RAxML version 7.2.7 withthe LG+G+F model conducted on CIPRES ScienceGateway Web server (on RAxML-HPC2 on Abe 7.2.7)[49]. For Bayesian posterior probabilities for branchnodes, we used MrBayes version 3.1.2 [50] on ParallelMrBayes Web Server at the BioHPC compute cluster atCBSU (http://biohpc.org/), with 1,713,600 generations,sampling trees every 100 generations, and discarding thefirst 5000 trees as a burnin. Convergence was evaluatedfrom running two independent chains. The effectivesample size was > 288.3 and 325.2, estimated by Tracerv1.5 [51]. Each of the four of individual protein data setswas analyzed with likelihood as explained above FigureS1a-S1d (in Additional file 1).Tree topology testsTo compare alternative phylogenetic positions for Olpi-dium, we used the Approximately Unbiased test and theweighted Shimodaira-Hasegawa test [52,53], both imple-mented by CONSEL v0.20 [54], using the site-wise like-lihood values estimated in PAUP v.4.0b10 [55]. Eachconstrained tree was based on an initial guide tree witha single internal branch, generated in MacClade version4.08 [56] or in Mesquite version 2.75 [57] (Additionalfile 6). The most likely tree (Additional file 6), giveneach constraint, was found using 50 search replicates inRAxML version 7.2.8 with the LG+G+F model con-ducted on CIPRES Science Gateway Web server (onRAxML-HPC2 on TeraGrid) [49].Additional materialAdditional file 1: Figure S1. The phylogeny of the kingdom Fungibased on likelihood analysis of amino acid sequences from singlegenes. Figure S1a, elongation factor 2 tree; Figure S1b, RNApolymerase II largest subunit tree; Figure S1c, RNA polymerase IIsecond largest subunit tree; Figure S1d, actin tree.Additional file 2: Figure S2 and Table S1. Figure S2. The phylogenyof the kingdom Fungi, including Rozella allomycis, based onlikelihood analysis of amino acid sequences of four concatenatedprotein-encoding genes. Table S1. Tree topology tests showing thatmost of the alternative phylogenetic positions of Rozella could notbe rejected.Additional file 3: Table S2. Species sequenced in this study,including voucher numbers and primer sets used.Additional file 4: Table S3. Primers used in this study.Additional file 5: Table S4. GenBank accession numbers ofsequences used in this study.Additional file 6: The initial guide trees with a single internalbranch, and the most likely trees given the constraint, that we usedfor the tree topology tests in Table 1. All trees are in Newickformat.AcknowledgementsThis research was financially supported by the US National ScienceFoundation Systematics and Population Biology/Assembling the Tree of LifeProgram through the collaborative research grant titled ‘Assembling theFungal Tree of Life’ (DEB-0732984), and through a Natural Sciences andEngineering Research Council of Canada Discovery grant to Berbee. Forcomputing support, we thank David Carmean (Simon Fraser University,Canada); Joseph Spatafora and Christopher Sullivan (Oregon State University,USA); and the Oregon State University Center for Genome Research andBiocomputing. The cultures were kindly supplied by the followingresearchers and culture collections; Kerry O’Donnell (NCAUR-ARS-USDA, USA),Joyce E. Longcore (University of Maine, USA), Peter M. Letcher (University ofAlabama, USA), UC Berkeley Microgarden (USA), Canadian Collection ofFungal Cultures (CCFC, Canada), Sammlung von Algenkulturen Göttingen(SAG, Germany), and UBC Herbarium, University of British Columbia. Authorsthank Bernard Ball (Lutzoni Lab, Duke University, USA) for sharingunpublished Ef-2 gene primer sequences.Author details1Department of Botany, 3529-6270 University Boulevard, University of BritishColumbia, Vancouver, British Columbia, V6T 1Z4 Canada. 2Agriculture andAgri-Food Canada, Pacific Agri-Food Research Centre, Summerland, BritishColumbia, V0H 1Z0 Canada. 3Department of Biological Sciences, TheUniversity of Alabama, Tuscaloosa, AL 35487, USA. 4Department of Biology,University of Victoria, P.O. Box 3020 - Station CSC, Victoria, British Columbia,V8W 3N5 Canada.Authors’ contributionsSS and JMD sequenced Ef-2 gene sequences. SS sequenced RPB1 and RPB2gene sequences. JEL sequenced and constructed actin data set. SSconstructed Ef-2, RPB1, and RPB2 dataset, and prepared the manuscript. DRprepared and provided DNA and RNA extracts from two Olpidium species.SS and MLB conducted the phylogenetic analysis. MLB participated in thedesign of study. All authors joined in reading, editing and approving themanuscript.Sekimoto et al. BMC Evolutionary Biology 2011, 11:331http://www.biomedcentral.com/1471-2148/11/331Page 8 of 10Received: 28 April 2011 Accepted: 15 November 2011Published: 15 November 2011References1. Ruiz-Trillo I, Burger G, Holland PWH, King N, Lang BF, Roger AJ, Gray MW:The origins of multicellularity: a multi-taxon genome initiative. Trends inGenetics 2007, 23(3):113-118.2. Stajich JE, Berbee ML, Blackwell M, Hibbett DS, James TY, Spatafora JW,Taylor JW: The Fungi. Current Biology 2009, 19(18):R840-R845.3. Wainright PO, Hinkle G, Sogin ML, Stickel SK: Monophyletic origins of theMetazoa: an evolutionary link with Fungi. 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Maddison DR, Maddison WP: MacClade. Sunderland: Sinauer;, 4.08 2005[http://macclade.org/macclade.html].57. Maddison WP, Maddison DR: Mesquite: a modular system forevolutionary analysis., 2.75 2011 [http://mesquiteproject.org].doi:10.1186/1471-2148-11-331Cite this article as: Sekimoto et al.: A multigene phylogeny of Olpidiumand its implications for early fungal evolution. BMC Evolutionary Biology2011 11:331.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitSekimoto et al. BMC Evolutionary Biology 2011, 11:331http://www.biomedcentral.com/1471-2148/11/331Page 10 of 10


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