Host plant specialization governed by facultative symbiont.

Materials and methods Samples of A. pisum were collected from natural populations on vetch or white clover, preserved in acetone, and subjected to DNA extraction individually. At each locality, 20 aphids were subjected to specific PCR detection of the endosymbionts as described (S1). The aphid strain TUt, naturally infected with PAUS, was derived from a parthenogenetic female collected on white clover at Tsuchiura, central Japan, in 2001 (S1). The stock culture of the strain was maintained on seedlings of the broad bean, Vicia faba, at 20C in a long day regimen (16L: 8D). Selective elimination and artificial transfer of PAUS were performed as described (S2). After the elimination/re-introduction of PAUS, the aphid strains were reared on seedlings of V. faba for 10 generations, which should eliminate possible side effects of the treatments. Subsequently, the aphids were transferred to either vetch or white clover, and cultured on each plant at least for 3 generations after the transfer. Vetch and white clover were grown from naturally-collected seeds in medium nutrient compost and used as pre-flowering plants at 3-4 weeks post-sowing. Three plants were potted in each container. To minimize winged morph production, a limited number of aphids (ca. 10) were maintained in each container. Each experiment was initiated by allowing an adult to larviposit on the plant for 6 hrs on the second day of reproduction. The newborn nymphs were defined as 0 day old, and reared individually on each of the host plants. Fitness parameters of the experimental aphids were monitored every 2 days. The experimental aphids were transferred to a fresh plant every 8 days, and their offspring were removed every 2 days. These experiments were conducted at 20C in the long day regimen (16L: 8D).