Materials and
methods Plant and seed material To test the effect of infection, host plant origin, and environmental factors (water and nutrient treatments), in August 2005, we collected seeds from multiple natural tall fescue populations by the Baltic Sea in localities that were geographically separated from selleck each other by approximately 500 km. These were the island of Åland (8 populations), the island of Gotland (9 populations), and the west coast of Sweden (6 populations). 10 to 50 individuals were collected from each population, and three seeds from each plant individual were stained for microscopic examination of the endophyte infection status (Saha et al. 1988). Neotyphodium coenophialum infectivity varied between 85–100% in all tall fescue populations from the three locations. Uninfected (E-) and infected (E+) seeds were combined separately from populations within each of the three study areas (Åland, Gotland, and coastal Sweden). In
other words, we pooled all E- seeds and then all E+ from the populations within each location to create three batches of E- seeds and three batches of E+ seeds that represented the three geographic origins. In addition to plants from natural tall fescue populations, we used E+ and E- K-31 (from T. MK-1775 mw Phillips, University of Kentucky) cultivar seeds in our experiment. To test the role of the endophyte on invertebrate communities while controlling for plant genotypic background, we N-acetylglucosamine-1-phosphate transferase experimentally removed the endophyte from portion of E+ seeds (manipulatively endophyte-free plants = ME-). To kill the fungus while the seeds remained viable, the E+ seeds were heat–treated by keeping the seeds in warm water (56-57°C) for 10–20 min. All tall fescue seeds from natural populations, K-31 cultivar and endophyte-removed seeds were germinated on moist tissue paper in Petri-dishes in a greenhouse and planted 7 days after germination to individual
pots with sand and peat mixture. Field experiment To test the role of endophyte infection, plant geographic origin and environmental factors, a common garden field experiment was established at Botanical Garden, University of Turku, Finland in 2004. The study site is at the edge of the northern distribution range of natural tall fescue populations and has been in cultivation in the past. It was tilled in the summer 2004 without nutrient Compound C mouse application. The experimental area was fenced to prevent large vertebrates (e.g., rabbits, deer) from browsing the plants. However, smaller vertebrates (e.g., voles) and invertebrates were allowed to freely access the area. The space between experimental plants was either mowed, hand weeded or sprayed with herbicide two times during the growing season to prevent interspecific competition in the field.