To determine whether specific miRNAs also change in expression, we counted the Illumina reads in samples of RNA pooled from the auditory forebrain of birds either 30 min after onset of song playback (Song group) or from matched controls (Silence group).
The stimulus sequence was presented in two manners: forward playback (F group: 1, 2, 3, 4, 5, 6…; the numbers indicate the frames of the original sequence) and reverse playback (R group: 3,600, 3,599, 3,598, 3,597, 3,596, 3,595…; Online Resource 6).
Although playback experiments in group-living species usually measure the response of a single subject at a time, an unknown number of group members are likely to perceive the playback stimulus as well.
The video packets with the same playback deadlines are grouped together to form GPs. Assume that the order of the OBUs in the service list is OBU 3, OBU 1, OBU 4, and OBU 2, which is decided by their scheduling weight factors (4).
Thus, each growl was used two times for playbacks within each group, except in the Cat-Growl group.
We tested whether the number of different song types sung in response to the playback differed between groups (own/unfamiliar song) by using unpaired t-test.
Ravens separated for up to three years responded differently to playbacks of former group members according to the categorization into affiliated versus nonaffiliated and familiar versus unfamiliar individuals.
In each subqueue, the packets with the same playback deadline are further grouped into a group of packets (GP).
This only occurred once and the subject was immediately released to rejoin her group, before the playback occurred.
Here we show that adult, pair-housed ravens not only respond differently to the playback of calls from previous group members and unfamiliar conspecifics but also discriminate between familiar birds according to the relationship valence they had to those subjects up to three years ago as subadult nonbreeders.
