Premating isolation and fecundity
We measure premating isolation among strains of the same subspecies (i.e., G×P; S×U) as well as different subspecies (i.e., G×U; P×S) using multiple‐choice mating experiments (Jennings et al. 2011). Virgin females and naïve males from different strains were allowed to feed in either red or blue colored food overnight and then placed together in bottles containing CYMA food supplemented with yeast in groups of 30 males and females per strain. Mating pairs were observed until half of all possible matings had occurred but for no longer than an hour, mating pairs were removed and identified based on the color of their abdomen (Gilbert & Starmer, 1985; Casares et al ., 1998). The experiments were run over 3-4 replicates (different days) of each cross. Data was pooled over replicates and the index of sexual isolation, IPSI, was calculated using the program JMating (Rolán‐Alvarez & Caballero, 2000). Positive Ipsi values are indicative of positive assortative mating and suggest premating isolation. We also estimated the asymmetry index (IAPSI(AB/BA)), which measures possible asymmetric differences in female preference for heterotypic males. Statistically significant deviations from random mating (i.e. IPSI= 0) were determined by bootstrapping 10,000 in JMating.
We tested fecundity of crosses between individuals of the same population as well as between individuals of different subspecies. We followed a protocol described in Gomes and Civetta (2014). Briefly, five 5 to 6 days-old naïve males and virgin females were placed together for 48 h in a vial containing CYMA food. Males were removed after 48 h and females transferred to a fresh vial five days after the initial set-up. Females were discarded after 5 days and progeny counted from both vials 23 days after the initial set-up. Each cross was replicated at least 5 times.