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Background: Inappropriate taxon definitions may have severe consequences in many areas. For instance, biologically sensible species delimitation of plant pathogens is crucial for measures such as plant protection or biological control and for comparative studies involving model organisms. However, delimiting species is challenging in the case of organisms for which often only molecular data are available, such as prokaryotes, fungi, and many unicellular eukaryotes. Even in the case of organisms with well-established morphological characteristics, molecular taxonomy is often necessary to emend current taxonomic concepts and to analyze DNA sequences directly sampled from the environment. Typically, for this purpose clustering approaches to delineate molecular operational taxonomic units have been applied using arbitrary choices regarding the distance threshold values, and the clustering algorithms., Methodology: Here, we report on a clustering optimization method to establish a molecular taxonomy of Peronospora based on ITS nrDNA sequences. Peronospora is the largest genus within the downy mildews, which are obligate parasites of higher plants, and includes various economically important pathogens. The method determines the distance function and clustering setting that result in an optimal agreement with selected reference data. Optimization was based on both taxonomy-based and host-based reference information, yielding the same outcome. Resampling and permutation methods indicate that the method is robust regarding taxon sampling and errors in the reference data. Tests with newly obtained ITS sequences demonstrate the use of the re-classified dataset in molecular identification of downy mildews., Conclusions: A corrected taxonomy is provided for all Peronospora ITS sequences contained in public databases. Clustering optimization appears to be broadly applicable in automated, sequence-based taxonomy. The method connects traditional and modern taxonomic disciplines by specifically addressing the issue of how to optimally account for both traditional species concepts and genetic divergence.

Species concepts are a notoriously difficult taxonomic problem in plant-parasitic fungal-like organisms such as downy mildews (Peronosporomycetes, Peronosporales). This is particularly evident in the largest downy mildew genus, Peronospora, which contains a number of economically important pathogens. Here, we investigate relationships of Peronospora species infecting Fabaceae (angiosperms, Rosidae) originating from various collections from different species of host plants and from different European locations by molecular phylogenetic analysis of ITS sequences. Molecular trees were inferred with ML, MP and Bayesian methods and rooted with Pseudoperonospora. As in other downy mildew groups, molecular data mainly support the use of narrow species delimitations and host range as a taxonomic marker. Fabaceae parasites appear to be subdivided into a number of lineages displaying a considerable degree of host specialization with respect to host genera, as well as host subgenera or species. The number of repeats of a repetitive part of the ITS1 is, within limits, characteristic of subgroups within the cluster of Trifolium parasites. We reveal new hosts for Peronospora found on the Iberian Peninsula.