Botanophila flies on Epichloë host species in Europe and North America: no evidence for co-evolution

Species of Epichloë (Ascomycota: Clavicipitaceae), which infect grasses, maintain an obligate symbiotic relationship with flies of the genus Botanophila (Diptera: Anthomyiidae). Sexual reproduction of the fungus usually requires a visit by Botanophila, which serves to ‘pollinate’ the fungus with spermatia of the opposite mating type; the flies in turn deposit their eggs on fungal tissues upon which the larvae feed. For a molecular phylogenetic study, a total of 108 fly larvae were collected from 10 different Epichloë species on various grasses in Europe and North America. Sequence analysis of the mitochondrial cytochrome oxidase gene (COII) detected six distinct Botanophila taxa that were associated with Epichloë. Three were restricted to samples from Europe, two to samples from North America, and one was present on both continents. In North America the common taxon identified as Botanophila lobata (Collin)] was found on Epichloë hosts of native grasses and on Epichloë typhina (Pers.: Fr.) Tul. and Epichloë festucae Leuchtm., Schardl and M.R. Siegel of the introduced grasses Dactylis glomerata L. and Festuca rubra L., respectively. In a maximum likelihood phylogram, one of the American taxa was most basal, while the other was nested within the European taxa, suggesting that European and American taxa of Epichloë-associated Botanophila spp. may not have separate origins. Analyses of the fly populations further indicated that up to four different Botanophila taxa can be present at a single location. On taxon level there was no specificity of flies for Epichloë host species (except taxon 6 that was found only on Epichloë glyceriae Schardl & Leuchtmann), although several taxa appeared to prefer some hosts over others. Comparison of molecular phylogenetic relationships of Botanophila species with those of the associated Epichloë hosts did not suggest co-evolution of the fungus and the fly. Thus, associations between Botanophila flies and Epichloë hosts may have arisen independently more than once.