PCR of plastid DNA and ITS sequences of figs

Plastid DNA, including six non-coding regions, the rps16 intron, trnG intron, petB intron, trnL intron, trnL-trnF spacer region, and the atpB-rbcL spacer region were amplified from a total of 418 samples of three fig species (i.e., F. erecta, F. formosana, and F. vaccinioides) (Table 1 and Supplementary Data 1).

Since the pollinator species B. silvestriana appeared to be associated with five distinct fig species in S. China and Hong Kong, we sought to investigate whether there was any host specificity between B. silvestriana and the five fig species. We specifically examined whether pollinator wasps could enter the receptive syconia of a fig species different from the species in which they were reared. We did this by comparing the ITS sequences of fig leaves and pollen carried by foundresses. The ITS sequences were amplified from 18 leaf DNA samples of four fig species (F. erecta, F. formosana, F. pyriformis, and F. abelii), and pollen DNA samples from three foundresses (one from F. pyriformis and two from F. formosana) (Supplementary Fig. 3).

PCR amplification was performed using LA-Taq (TaKaRa) or KOD-Plus (TOYOBO) DNA polymerase in 25 μL reaction mixtures containing 1 μL (figs) or 2 μL (pollinator wasps) of template DNA and following the procedures outlined in previous studies^16,24,32,53 with some modifications. The PCR primers and conditions are shown in Supplementary Tables 3 and 4, respectively.