Date Published: March 12, 2018
Publisher: Public Library of Science
Author(s): Tyler B. Bourret, Robin A. Choudhury, Heather K. Mehl, Cheryl L. Blomquist, Neil McRoberts, David M. Rizzo, Mark Gijzen.
Phylogenetic relationships between thirteen species of downy mildew and 103 species of Phytophthora (plant-pathogenic oomycetes) were investigated with two nuclear and four mitochondrial loci, using several likelihood-based approaches. Three Phytophthora taxa and all downy mildew taxa were excluded from the previously recognized subgeneric clades of Phytophthora, though all were strongly supported within the paraphyletic genus. Downy mildews appear to be polyphyletic, with graminicolous downy mildews (GDM), brassicolous downy mildews (BDM) and downy mildews with colored conidia (DMCC) forming a clade with the previously unplaced Phytophthora taxon totara; downy mildews with pyriform haustoria (DMPH) were placed in their own clade with affinities to the obligate biotrophic P. cyperi. Results suggest the recognition of four additional clades within Phytophthora, but few relationships between clades could be resolved. Trees containing all twenty extant downy mildew genera were produced by adding partial coverage of seventeen additional downy mildew taxa; these trees supported the monophyly of the BDMs, DMCCs and DMPHs but suggested that the GDMs are paraphyletic in respect to the BDMs or polyphyletic. Incongruence between nuclear-only and mitochondrial-only trees suggests introgression may have occurred between several clades, particularly those containing biotrophs, questioning whether obligate biotrophic parasitism and other traits with polyphyletic distributions arose independently or were horizontally transferred. Phylogenetic approaches may be limited in their ability to resolve some of the complex relationships between the “subgeneric” clades of Phytophthora, which include twenty downy mildew genera and hundreds of species.
Oomycetes (Phylum Oomycota, Kingdom Straminipila) are eukaryotic microbes that include many important pathogens of plants . In particular, species in the genus Phytophthora are among the greatest socioeconomic threats to agriculture and natural ecosystems [2,3]. Also important is the group of oomycetes known as the downy mildews, which comprise twenty genera and a large portion of described oomycete species [4,5]. Phytophthora and the downy mildews, with the early-diverging Halophytophthora, Nothophytophthora and Phytopythium constitute a monophyletic clade known as the Peronosporaceae s.l. [1,4,6]. Nearly every member of the Peronosporaceae is associated with plants, either as a parasite (necrotrophic, biotrophic or hemibiotrophic) or as a saprotroph of dead plant tissue.
The alignment files and tree output files for all analyses can be found at TreeBASE.org under accession S21585. The results of the phylogenetic inference from the 118-taxon data set are illustrated in Fig 1. Most bipartitions were significantly supported in all analyses, including strong support for each of the subgeneric clades of Phytophthora. Deeper branching patterns between the clades were not well-supported and often varied between analyses. Exceptions included strong support for Phytophthora+downy mildews, Phytophthora clades 1–8,12–14+downy mildews, and Phytophthora clades 1–5,12–14+downy mildews. Although the eleven included previously recognized subgeneric clades of Phytophthora were recovered with strong support, the results suggest at least four additional clades should be recognized to account for the downy mildews and three Phytophthora taxa that could not be placed into the other clades. These clades were provisionally given numbers 13–16 (Fig 1). The downy mildews were found not to be monophyletic; DMPHs formed a separate clade, while DMCCs, Sclerospora (representing GDMs) and BDMs were placed in a strongly supported clade with P. taxon totara. Phytophthora clade 1, DMPHs (clade 16), P. cyperi (clade 14), P. quercina/ohioensis (clade 12) and P. taxon mugwort (clade 13) often clustered, but relationships were not consistent or consistently well-supported; there was a similar association between Phytophthora clade 5 and P. taxon totara+DMCC+GDM+BDM (clade 15). The trees inferred from the larger 135-taxon alignment were largely congruent with the first (Fig 2). The remaining six genera of DMPH formed a monophyletic clade with Bremia and Plasmopara. No significant support was found for the early diverging branches of the DMPH or the GDM and GDM were paraphyletic with respect to BDM. In the MrBayes analysis (not illustrated, available at TreeBASE S21585), Baobabopsis formed a clade with Phytophthora clade 12 rather than with the rest of the GDMs and BDMs; this contrasted the topology in the most likely tree inferred by Garli (Fig 2), where Baobabopsis clustered with the other GDMs. The same placement of Baobabopsis away from the other GDMs was also seen in the MrBayes analysis of the mitochondrial-only tree derived from the 135-taxon data set (S3 Fig).
Although no evolutionary scenarios can be ruled out, our analysis suggests multiple origins of obligate biotrophy within the Phytophthora para-genus, with the most parsimonious scenario suggesting two independent origins: once in the lineage leading to the BDMs, DMCCs and GDMs, and a second time in the lineage leading to P. cyperi and the DMPHs (Fig 2). Only Göker and Stamatakis  and McCarthy and Fitzpatrick  found the same separation of DMs into two groups, with the DMPHs apart from the other downy mildews, although relatively few prior Phytophthora+downy mildew phylogenies included the DMPHs (Table 1). The close and well-supported relationship between the BDMs, DMCCs and GDMs with the southern hemisphere, foliar, conifer-associated Phytophthora taxon totara  is notable. Although downy mildews arising from a foliar Phytophthora ancestor presents a reasonable scenario, none has yet been found on a non-angiosperm host . The topology of the BDM and GDM (Fig 2), with paraphyletic GDM suggests BDM are the result of a host jump from a graminicolous ancestor. While GDM appear confined to Poaceae hosts [4,54], Hyaloperonospora has diversified onto non-Brassicaceae hosts . The topology of the DMPH suggests the common ancestor had specialized on the Asteraceae, with host jumps to the Ranunculaceae by ancestors of Plasmoverna and jumps to other eudicots by some members of Plasmopara.
Relationships within the Peronosporaceae s.l. remain enigmatic. This study highlights the importance of adding sequences from previously unsampled taxa, which may have a greater impact on difficult phylogenies than adding additional loci . The 135-taxon data set produced the first phylogenies to contain all currently recognized downy mildew genera, a significant contribution to the overall understanding of downy mildew evolution. According to our findings, two of the four downy mildew groups are not monophyletic, one arising from within the other. The diversity of taxa included in the data set illuminated several previously unrecognized, strongly supported subgeneric clades within Phytophthora s.l.; two clades contained downy mildew taxa and one contained both Phytophthora and downy mildews. Incorporating an obligate biotrophic Phytophthora species for the first time, the trait appears to have evolved at least twice within the Peronosporaceae, and, owing to the obligate biotrophic Albuginales, at least three times within the Peronosporomycetes. As this manuscript was being prepared, preliminary phylogenetic placement of Phytophthora polygoni suggests the existence of an additional, uncharacterized subgeneric clade and an additional acquisition of obligate biotrophy within Phytophthora s.l.