Females commonly prefer directed songs more than undirected songs.

<p>Among the birds that expressed a significant preference in tests of directed versus undirected song, all preferred directed song. Among birds that had experience with the male singers (left, birds 1-10), 5 expressed a significant preference for directed song (chi-squared test, p < 0.05, filled symbols) and 5 expressed no significant preference (p ≥ 0.05, open symbols) (square indicates bird in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089438#pone-0089438-g004" target="_blank">Figure 4A</a>, triangle indicates bird in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089438#pone-0089438-g004" target="_blank">Figure 4B</a>). Among birds that did not have experience with the male singers (right, birds 11-18), 5 birds expressed a significant preference for directed song and 3 birds expressed no significant preference (open and filled symbols as in birds 1-10; star indicates birds in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089438#pone-0089438-g004" target="_blank">Figure 4D</a>, diamond indicates bird in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089438#pone-0089438-g004" target="_blank">Figure 4E</a>). Preference for directed song was evident across different males but was ultimately specific to each female, as the 10 significant responses (filled symbols) correspond to 5 different males, but not all females that heard those songs preferred directed song.</p

Overview of experimental methods and flow.

<p>In each Phase, we played song stimuli to female BFs in the absence of a male bird and tallied the number of responses to each stimulus. (A) In Phase 1, we investigated the degree to which calls as as reliable as CSDs as an index of female BF mate preference. In one group, females were tested first with no implant and then after a minimally invasive subcutaneous estradiol implant (N = 6 birds). In a second group, females were tested first with an estradiol implant and then after recovery from implant removal (N = 6 birds). In a third group, females were implanted with a subcutaneous sham implant (N = 4 birds). None of the birds tested in Phase 1 were also used in Phase 2. (B) In Phase 2A, we investigated the degree to which female BFs are consistent in their song preference across time and trials. In one group (“experienced”), females had interacted with the male birds from which song stimuli were recorded (N = 10 birds). In another group of birds (“inexperienced”), females had never interacted with those males (N = 11 birds). After a bird had completed Phase 2A, it moved on to Phase 2B in which we created stimuli from each female’s most-preferred male and tested the degree to which female birds prefer songs performed in the presence of a female (“directed”) versus songs performed when the male is alone (“undirected”). Three inexperienced birds failed to meet the criteria for inclusion in the results of Phase 2.</p

Parameters of Directed and Undirected Songs of Each Male Used in these Experiments

<p>Parameters of Directed and Undirected Songs of Each Male Used in these Experiments</p

Individual females vary in their selectivity for songs of different males.

<p>Individual females expressed a range of selectivity for the songs of individual males, and the nature of those preferences was not different between birds that did (panels A-C) or did not (panels D-F) have previous experience with the associated male singers. (A) Among birds with experience of the male singers, some females were very selective for the song(s) of individual males, such as the female shown here (selectivity index  =  3.76; points indicate mean; lines indicate SE in panels A, B, D, E). (B) Other females were not selective, responding similarly to the songs of many males, as in the case of the female shown here (selectivity index  =  1.43). (C) Across all 11 birds that had experience with the male singers, selectivity indices ranged from 1.43 to 3.97 (mean ± SE  =  2.65±0.31, dark solid lines indicate the birds shown in panels A (filled circles) and B (filled squares), dotted line indicates level of chance). (D) Among birds that did not have experience with the male singers, the response were very similar, with one bird expressing very selective responses (selectivity index  =  3.76) and (E) another bird responding much more broadly (selectivity index  =  1.89). (F) Across all 10 birds that did not have experience with the male singers, selectivity indices ranged from 1.60 to 3.76 (mean ± SE  =  2.47±0.17) and were indistinguishable from those detected for birds that had experience with the singers (statistics detailed in the text; dark solid lines indicate the birds shown in panels D (open circles) and E (open squares), dotted line indicates level of chance).</p

Females prefer songs of some males more than others.

<p>Females tended to prefer the songs of two males more than the other males tested here, and preferences for individual males were broadly similar between birds that did or did not have previous experience with the associated male singers. (A) Each female had one song for which she generated more calls than in response to any other song, which was deemed the most-preferred song type for that bird. Across all 21 birds tested, songs of two males (identified here as B and F) were more commonly preferred than the songs of other birds (filled symbols  =  10 females that had experience with the associated singers, open symbols  =  11 females that did not have experience with the associated singers). (B) When we expanded that consideration to include not just the top-ranked song for each bird but also the second-ranked song for each bird, that broad preference for males B and F was also evident (symbols as in panel A). Across the 21 birds tested here, 5 different songs were ranked as the most-preferred stimulus, and 6 different songs were ranked as either the top-ranked song or the second-ranked song. Across the population (N = 21 birds), male identity had a profound effect on female preference, but population preferences were similar regardless of whether the females did or did not have experience with the male singers (statistics detailed in the text).</p

Different facets of male song can be evaluated independently.

<p>When different facets of song selectivity are compared for each bird, the degree of selectivity for individual identity and the degree of selectivity for directed song are not related. That was true for both the group that had experience with the male singers (filled symbols; square indicates bird in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089438#pone-0089438-g004" target="_blank">Figure 4A</a>, triangle indicates bird in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089438#pone-0089438-g004" target="_blank">Figure 4B</a>) and females that had no experience with the male singers (open symbols; star indicates birds in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089438#pone-0089438-g004" target="_blank">Figure 4D</a>, diamond indicates bird in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089438#pone-0089438-g004" target="_blank">Figure 4E</a>; statistics detailed in the text). Thus, an individual bird can be selective for one aspect of male song but unselective for another aspect.</p

Calls are an index of female BF mate choice.

<p>Each female is represented by 3 data points (N = 12 birds, some points overlap) indicating the magnitude of its response to its most-preferred song (filled circles), its least-preferred song (open squares), and a randomly selected song of intermediate subjective value (filled triangles). Each of the three responses from each bird have been normalized to the total number of responses performed by that bird in response to all stimuli, so data are plotted as the percent of all CSDs (x-axis) and all calls (y-axis) that each bird performed in response to all stimuli. These data reveal good agreement between CSDs and calls (statistics detailed in the text), indicating that calls are a valid index of adult female BF mate choice.</p