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Delineating operational taxonomic units (OTUs) is a central element in any culture-independent analysis of environmental microbial eukaryotic diversity. Previous studies either have not justified their choice in sequence distance used to bin small-subunit ribosomal RNA (SSU rRNA) gene sequences amplified from environmental samples into OTUs, or have used a value based on the average across a broad sampling of microbial eukaryotes. Here, we analyse distances (320 922 pairwise comparisons) among sequences just from identified ciliates, and compare these with their taxonomic hierarchy. Our results show that no single sequence similarity value can always and unambiguously delineate species boundaries and higher taxa. Nevertheless, we suggest the use of 98% similarity to delineate ciliate OTUs because this threshold at least accounts for intra-specific polymorphism among multiple rRNA cistron copies. However, we suggest refraining from reconciling SSU rRNA gene-based OTUs and ciliate morphotypes; these OTUs should be used to analyse ciliate phylotype diversity, not ciliate species diversity.

Fungal colonization can significantly affect the secondary metabolism of the host plants. We tested the impact of a common below-ground symbiosis, i.e., ectomycorrhiza formation, on poplar leaf chemical components that are involved in the defence against a common disease, i.e., rust fungi, in N-deficient soil. A rust-susceptible poplar clone (Populus trichocarpa x deltoides 'Beaupre') was (a) non-associated with ectomycorrhizal fungus (EM) Hebeloma mesophaeum (Pers.) Quelet MUN and non-infected with rust fungus Melampsora larici-populina Kleb. (isolate 98AG31), (b) associated with EM, (c) inoculated with rust fungus and (d) associated with EM and inoculated with rust fungus. Poplar leaves were analysed by photometric and mass spectrometric techniques (liquid chromatography-tandem mass spectrometry (LC-MS/MS), pyrolysis-field ionization mass spectrometry (Py-FIMS)). Both rust infection and mycorrhiza formation led to increased proportions of condensed tannins in relation to total phenolics (13% in the control, 18-19% in the fungal treatments). In contrast, salicylic acid concentration (6.8 mu g g(-1) in the control) was higher only in the rust treatments (17.9 and 25.4 mu g g(-1) with rust infection). The Py-FIMS analysis revealed that the rust-infected treatments were significantly separated from the non-rust-infected treatments on the basis of six flavonoids and one lipid. The relative abundance of these components, which have known functions in plant defence, was decreased after rust infection of non-mycorrhizal plants, but not in mycorrhizal plants. The results indicate that the ectomycorrhizal formation compensated the rust infection by a decrease in the flavonoid syntheses. The study provides new evidence for an interactive response of mycorrhizal colonization and infection with rust fungi in the metabolism of poplar.