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.