Female Choice Assay:
Prior to the mate choice assay, each female’s cage was moved into a sound attenuation chamber equipped with one audio speaker. Females were given 24 hours to habituate to the chambers, at which point operant perches were installed on either side of the front cage door, so that total perches in the cage included two operant perches and two normal perches placed diagonally in the back of the cage. Operant perches were placed approximately 10 cm above the cage floor and 25 cm apart. Perches were approximately 9 cm long, ½ cm in diameter, and made of wood. Perches were attached to Honeywell lever arm depression micro-switches. The perch on the left when facing the cage was designated perch A, and the perch on the right was designated perch B. Hopping on a perch caused the perch to lower slightly and trigger a pressure-sensitive button on the micro-switch, which in turn triggered playback of a song through the speaker. In this type of operant conditioning, the song itself serves as the reward and encourages females to continue eliciting song through hops (Stevenson 1967; Riebel 2000; Anderson 2009). All songs were volume-adjusted to 65 dB SPL at the chamber center. All operant data were collected through Sound Analysis Pro (see Tchernichovski et al. 2000).
Females learned to trigger song during an initial training period (Anderson et al. 2014). For training, females were presented with conspecific/ heterospecific song pairs (heterospecific song was from rufous-collared sparrows, Zonotrichia capensis ). Whether perch A played conspecific and perch B played heterospecific, or vice versa, was alternated between birds, and switched every day for the same bird. Switching songs between perches helped control for side bias. In order to pass training, females had to hop on both perches for two days in a row. All females passed training within 4 days.
After training, females were presented with Solver/Non-Solver stimuli. Females were given a new pair of Solver and Non-Solver songs every day, in which a single song from a Solver male was triggered by a hop on one perch, and a single song from a Non-Solver male was triggered by a hop on another perch. The side that played Solver song was switched each day to control for side bias. Each bird heard 4 of 6 Solver/Non-Solver pairs, as female response tends to drop off after prolonged periods in preference testing. Testing multiple stimulus sets on each female also greatly reduces the risk that a chosen stimulus set is non-representative of its category and yet repeatedly measured (i.e. pseudoreplication in acoustic experiments; Kroodsma 1989). Trials ran from 11am to 5pm each day, during which the number of hops on each perch was recorded. Outside of trial times, hops on a perch did not trigger playback.