doi:10.5598/imafungus.2011.02.02.07 IMA FUNGUS · VOLUME 2 · No 2: 163–171 The inclusion of downy mildews in a multi-locus-dataset and its reanalysis ARTICLE reveals a high degree of paraphyly in Phytophthora Fabian Runge1, Sabine Telle2,3, Sebastian Ploch2,3, Elizabeth Savory4, Brad Day4, Rahul Sharma2,3,5, and Marco Thines2,3,5 1University of Hohenheim, Institute of Botany 210, D-70593 Stuttgart, Germany 2Biodiversity and Climate Research Centre (BiK-F), Senckenberganlage 25, D-60325 Frankfurt am Main, Germany; corresponding author e-mail: [email protected] 3Senckenberg Gesellschaft für Naturforschung, Senckenberganlage 25, D-60325 Frankfurt am Main, Germany 4Michigan State University, Department of Plant Pathology, 104 Center for Integrated Plant Systems, East Lansing, MI 48824, USA 5Goethe University Frankfurt am Main, Department of Biological Sciences, Institute of Ecology, Evolution and Diversity, Siesmayerstr. 70, D-60323 Frankfurt (Main), Germany Abstract: Pathogens belonging to the Oomycota, a group of heterokont, fungal-like organisms, are amongst the Key words: most notorious pathogens in agriculture. In particular, the obligate biotrophic downy mildews and the hemibiotrophic AU test members of the genus Phytophthora are responsible for a huge variety of destructive diseases, including sudden downy mildews oak death caused by P. ramorum, potato late blight caused by P. infestans, cucurbit downy mildew caused by multigene phylogeny Pseudoperonospora cubensis, and grape downy mildew caused by Plasmopara viticola. About 800 species of Peronosporaceae downy mildews and roughly 100 species of Phytophthora are currently accepted, and recent studies have revealed Phytophthora that these groups are closely related. However, the degree to which Phytophthora is paraphyletic and where taxonomy exactly the downy mildews insert into this genus in relation to other clades could not be inferred with certainty to date. Here we present a molecular phylogeny encompassing all clades of Phytophthora as represented in a multi- locus dataset and two representatives of the monophyletic downy mildews from divergent genera. Our results demonstrate that Phytophthora is at least six times paraphyletic with respect to the downy mildews. The downy mildew representatives are consistently nested within clade 4 (contains Phytophthora palmivora), which is placed sister to clade 1 (contains Phytophthora infestans). This finding would either necessitate placing all downy mildews and Phytopthora species in a single genus, either under the oldest generic name Peronospora or by conservation the later name Phytophthora, or the description of at least six new genera within Phytophthora. The complications of both options are discussed, and it is concluded that the latter is preferable, as it warrants fewer name changes and is more practical. Article info: Submitted 21 September 2011; Accepted 20 October 2011; Published 11 November 2011. INTRODUCTION Göker et al. 2003, 2007, Thines et al. 2008, 2009, Thines 2009). The close relationship of the downy mildews and Oomycetes are a group of organisms that superficially Phytophthora revealed by these studies is in contrast to the resemble fungi in their hyphal growth and absorptive way of widely used taxonomic classifications of Waterhouse (1973) nutrition. However, they are not closely related to Mycota, and Dick (1984, 2001), in which Phytophthora and Pythium but belong to a group of heterokont organisms, Straminipila were grouped together in the family Pythiaceae. Although (Dick 2001), which also includes diatoms and sea-weeds. Cooke et al. (2000) inferred a position of Peronospora sparsa Oomycetes have adapted to parasitism of plants at least three as a sister group of clade 4 (as defined in that study) based on times, once in the Saprolegniales in the genera Aphanomyces ITS sequences alone, no substantial phylogenetic resolution and Pachymetra (Riethmüller et al. 1999, Diéguez-Uribeondo was present on the phylogenetic backbone, thus failing to et al. 2009), and separately in Albuginales and Peronosporales position this group within the genus Phytophthora. Other (Riethmüller et al. 2002, Hudspeth et al. 2003, Thines et al. studies (including multi-locus studies) that included both downy 2008). While the evolution of obligate biotrophy seems to be mildew and Phytophthora species have so far not resolved the an ancient occurrence for the white blister rusts (Thines & placement of downy mildews in relation to the different clades Kamoun 2010), the downy mildews have more recently arisen of Phytophthora (Riethmüller et al. 2002, Göker et al. 2007, from Phytophthora-like ancestors (Riethmüller et al. 2002, Thines et al. 2009, Giresse et al. 2010). Additionally, Thines et © 2011 International Mycological Association You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights. VOLUME 2 · NO. 2 163 Runge et al. al. (2009) demonstrated that the support for the sister-group computer package v. 8 (Lasergene, Madison, WI), and were relationship of Peronospora and clade 4 inferred by Cooke et aligned separately using MAFFT v. 6.240 (Katoh et al. 2005) al. (2000) could have been the result of an alignment artefact. using a webserver interface (http://www.genome.jp/tools/ Conversely, a recent study by Blair et al. (2008) addressed the mafft/). The G-INS-i algorithm was chosen for all alignments. phylogenetic relationships of Phytophthora species with good Subsequently, the aligned sequences were concatenated ARTICLE resolution, but no downy mildew was included in that study, for phylogenetic analyses and no further editing was done leaving their placement to speculation. Downy mildews have on the alignment to ensure reproducibility and to prevent been shown to be a monophyletic assemblage by Göker et introduction of bias. After the removal of leading and trailing al. (2007). However, Göker & Stamatakis (2006) later (in spite gaps 6282 nucleotide sites were included in the phylogenetic of being published earlier than Göker et al. 2007) came to the analyses. These comprised seven loci: 1119 bp of the beta- conclusion that a placement of Phytophthora clade 1 within the tubulin gene, 493 bp of the 60S ribosomal protein L10 gene, downy mildews would also be possible, although no support 873 bp of the translation elongation factor 1-alpha gene, 720 could be obtained for this scenario. The question of which bp of the 28S nuclear ribosomal DNA gene, 646 bp of the is the sister clade of the downy mildews, and how this clade glyceraldehyde-3-phosphate dehydrogenase gene, 1438 bp is embedded among the different lineages of Phytophthora of the heat shock protein 90 gene, and 993 bp of the enolase therefore continues to be controversial, but is fundamental for gene. The alignment, together with the tree from the Bayesian understanding the evolution of this group of important plant Analysis shown in Fig. 1, has been deposited in TreeBASE pathogens, especially with respect to the evolution of biotrophy. (www.treebase.org) under the accession number S11829. In addition, the taxonomic status of many Phytophthora The general time reversible (GTR) model was selected for species depends on the degree of paraphyly of the genus. At the concatenated alignment using Modeltest v. 3.7 (Posada least with two clades, 9 and 10, Phytophthora is paraphyletic & Crandall 1998) and PAUP v. 4.0b10 (Swofford 2002), with with respect to downy mildews (Cooke et al. 2000, Göker et al. gamma-distributed substitution rates (shape parameter = 2007, Thines et al. 2009), but so far, the degree of paraphyly 0.69) and proportion of invariable sites (pinv = 0.54). The of Phytophthora could not be resolved. Therefore, it was values of these parameters were included in the Bayesian the aim of this study to resolve the phylogenetic placement and Minimum Evolution analyses. of the monophyletic downy mildews (represented by the two Minimum Evolution (ME) analysis was done using MEGA divergent downy mildew genera for which genome data are v. 4.0 (Tamura et al. 2007), with the gamma-distributed currently available) among Phytophthora and to test this substitution rates as inferred by Modeltest and using placement statistically, to further clarify the relationships within the Maximum-Composite-Likelihood substitution model. this group of important plant pathogens. For inferring tree robustness, 1000 bootstrap replicates (Felsenstein 1985) were computed. For Maximum Likelihood (ML) inference, the RAxML MatERIALS AND METHODS webserver at http://phylobench.vital-it.ch/raxml-bb/ (Stamatakis et al. 2008) was used with standard settings All sequences of Phytophthora and Pythium were obtained and maximum likelihood search, including an estimation of from the study of Blair et al. (2008) available in the National invariable sites. The analysis was repeated five times with Center for Biotechnology