Pleospora species with Stemphylium anamorphs: a four locus phylogeny resolves new lineages yet does not distinguish among species in the Pleospora herbarum clade.

Stemphylium is a genus of plant pathogens and saprobes in the Pleosporaceae (Pleosporales, Dothideomycetes, Ascomycetes). The teleomorphs of Stemphylium, where known, are in Pleospora, with Pleospora herbarum as the type. The goal of this study was to present a rigorous phylogenetic analysis of the relationships among Stemphylium isolates with particular emphasis on species delimitation in the P. herbarum clade, on possible new species and on the relationship of clades to cultures from type specimens. Our taxon sampling comprised 110 Stemphylium strains collected worldwide from various hosts and DNA sequences from four loci, from the ITS, the protein encoding GPD and EF-1 alpha genes and the intergenic spacer between vmaA and vpsA. A large EF-1 alpha intron delimited by noncanonical splice sites and encoding putative proteins was present in three unrelated isolates and was excluded from analyses. Isolates comprised 23 representatives derived from type strains, compared to type strains or otherwise connected to type material, 40 unnamed strains morphologically similar to the type P. herbarum, four strains from an outbreak of Stemphylium leaf blight of cotton in Brazil and eight strains collected in British Columbia mainly from nonagricultural hosts. Our findings provided strong support for the main groupings of Stemphylium obtained earlier and also revealed six possible new species. Other variation within morphological species might point to additional cryptic species. On the other hand, even with four loci, cultures ex-type of five species including P. herbarum were inseparable. We speculate that being self-fertile the clade including P. herbarum might represent a group of highly inbred, morphologically distinct lineages that have yet to accumulate detectable species-specific sequence variation. The lack of variation in P. herbarum clade contrasts with many other a priori defined morphological species where multigene phylogenetic analyses revealed new cryptic species.