Literature Review Detailed Summary

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I have 9 primary source article.

For each one I need a Literature Review Detailed Summary. which is provided in the files, only four question to answer for each article.

Each summary should be in separate page.

I uploaded only 4 of them, I will post the rest after that because there is no space to upload them

so 9 articles total will be.

The Singing Behavior of Female Northern Cardinals Author(s): Gary Ritchison Source: The Condor, Vol. 88, No. 2 (May, 1986), pp. 156-159 Published by: University of California Press on behalf of the Cooper Ornithological Society Stable URL: http://www.jstor.org/stable/1368910 . Accessed: 14/01/2014 12:28 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp . JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact support@jstor.org. . University of California Press and Cooper Ornithological Society are collaborating with JSTOR to digitize, preserve and extend access to The Condor. http://www.jstor.org This content downloaded from 157.89.111.52 on Tue, 14 Jan 2014 12:28:19 PM All use subject to JSTOR Terms and Conditions TheCondor88:156-159 Society 1986 ? The CooperOrnithological THE SINGING BEHAVIOROF FEMALENORTHERN CARDINALS1 GARYRITCHISON Departmentof BiologicalSciences,EasternKentuckyUniversity,Richmond,KY 40475 Abstract. Observationswere made and playbackexperimentswere performedin centralKentuckyin an attemptto determinethe function(s)of singingby femaleNorthernCardinals(Cardinalis cardinalis).Singingby femalecardinalswas uncommonand was noted primarilyduringthe period after males had establishedterritoriesand before nesting had begun (earlyMarchto mid-May). When singing,femalesweregenerallyjoined by singingmates. Experimentsrevealedthat females rarelysang in responseto playbackof male or female songs. Such evidence suggeststhat singing by female NorthernCardinalsis importantin pair bonding and, perhaps,in reproductivesynchronization. Key words: NorthernCardinals;Cardinaliscardinalis;singing;pair-bondmaintenance. INTRODUCTION Althoughfemalessingless thanmales,or not fledging of the young, it would suggest that at all, in mostoscinespecies,singingdoesap- singing might play some role in family-group pearto be a regularfeatureof femalebehavior maintenance and might not be important in in severalspecies(Ritchison1983a).Amongthe the formation of pair-bonds or in territorial speciesin whichsingingby femaleshas been defense.Playbackexperimentswereused in an reportedis the NorthernCardinal(Cardinalis attempt to further clarify function. For excardinalis).For example,Bent (1968:11)in- ample, an increasedrate of singingin response dicatedthat in the NorthernCardinal"both to playbackof conspecificsong within the tersexes sing,and the song of the femaleis but ritory might indicate that female song serves littleinferiorto thatofthemale."Lemon(1965) a territorialfunction (Beletsky 1983). Further, noted that both male and female Northern if males, whose singing is known to serve in of severaldis- territorialdefense,singin responseto playback Cardinals possesseda repertoire tinctivesongs. and females do not, this would suggest that Onlya fewpossiblefunctionshavebeensug- singingmightservedifferentfunctionsin males gestedfor singingby females.Beletsky(1982) and in females. thatsingingby femaleNorthernOrisuggested oles (Icterusgalbula)playssome rolein pair- METHODSAND MATERIALS bond maintenance.Ritchison (1983a) pro- Field work was undertaken from January posed that singingby female Black-headed throughSeptemberin 1983 and 1984 and from was mid-FebruarythroughearlySeptemberin 1985 Grosbeaks(Pheucticusmelanocephalus) of importancein family-groupmaintenance. at the CentralKentuckyWildlifeManagement Beletsky(1983) reportedthat female Red- Area, located 17 km southeast of Richmond, wingedBlackbirds(Agelaiusphoeniceus)pos- Madison County, Kentucky. Observations sessedtwo songtypes,one of whichprobably were made at least three times weekly. Obserfunctionsin pair-bondmaintenanceand the vation periodswere at least two hr in duration defense.Ina reviewof avian and began shortlyaftersunrise.I attemptedto otherin territorial vocalbehavior,Nottebohm(1975) suggested record on tape all observed bouts of female thatsingingby femalesmayplaysomerolein song. Whetherrecordedor not, for each bout pair formationor may aid in territorialde- I noted the identity and location of the female, fense.The objectiveof the presentstudywas the number of songs uttered duringthe bout, to ascertainthe possiblefunction(s)of female and the location (if known)and behavior(singing or not singing)of the nearestmale. I define songin the NorthernCardinal. I usedbothfieldobservationsandplayback a "bout"as a series of songs separatedin time in aneffortto determinethefunc- from each other by intervals that were signifexperiments tion of femalesongin the NorthernCardinal. icantlylongerthan the intervalsbetweensongs Observationswere undertakento determine within a bout (Farabaugh1982). During the whenandin whatsituationsfemalecardinals three years of the study I monitored fifteen sang.Such informationcould providesome pairs of cardinals.All cardinals under obserinsightinto function.For example,if singing vation were capturedin mist nets and marked werelimitedto theperiodjustbeforeandafter with coloredleg bandsand coloredplastictape attachedto the rectrices(Ritchison 1984). Re' Received 10 December1984. Finalacceptance21 Oc- cordingswere made with a Uher Report Monitor tape recorderwith a Dan Gibson parabolic tober 1985. [1561 This content downloaded from 157.89.111.52 on Tue, 14 Jan 2014 12:28:19 PM All use subject to JSTOR Terms and Conditions SINGING OF FEMALENORTHERN CARDINALS reflectorand microphone.Playbacktapes were subsequentlymade from these recordings. Playbackexperimentswith six females were conductedfrom 11 April to 20 May 1983 and with six more females from 23 April to 1 May 1985. Each playbackexperiment consisted of three 5-min segments(pre-test,test, post-test). In 1983 each bird was tested twice with the songs of a neighboringmale and twice with the songs of a neighboringfemale. In 1985 each female was tested twice with the songs of a non-neighboringfemale; in addition, a study skin of a female NorthernCardinalwas placed within 1 m of the loudspeaker.Birdswith common territorial boundaries were designated neighbors. For each test a loudspeaker was placed 2 to 3 m above the ground in a bush or treenearthe centerof the territory.Different tests with individual females were at least two days apart. All experiments were conducted between 0700 and 1200. The followingfeaturesof responsewereused to ascertaina bird's reaction to playback:(1) Numberof songs and notesper song. Songs of NorthernCardinalsmay vary considerablyin the number of notes per song and previous studies suggesta possible correlationbetween the numberof notes per song and a bird'slevel of aggression(Gottfried and Gottfried 1978). Notes were utteredby test birds at a rate that allowedaccuratecounting.(2) Distanceofclosest approach to the speaker. (3) Number of flights. Flights of less than 50 cm were not counted. (4) Number of chip calls. Northern Cardinals utter these calls in various situations, and they may indicateexcitement.Lemon (1968a) reportedthat territorialmales utteredchipcallsin responseto trespassingmales and females. The Wilcoxon signed-rankstest was used for all statisticalcomparisons(Sokal and Rohlf 1969). RESULTS FIELDOBSERVATIONS The singingbehavior of female NorthernCardinals differed from that of males in several respects.First,femalessangless frequentlythan males. I spent over 45,000 min in the field and noted only 207 min of female song (less than 0.5%of the time). In addition,male and female Northern Cardinalsdiffered in the timing of their singing. Whereasmales begin singing in January or February(Laskey 1944), females did not begin singinguntil March(Fig. 1). Further,whereasmales continuedsinginguntillate Augustor earlySeptember(Fig. 1;Laskey1944, Lemon 1967), I rarelyheard singingby female cardinals after mid-May (Fig. 1). Most pairs of cardinalsbegan nestingby late April. Thus, 150 601 d' 501 -85 125 I 0 40- 100 75 30Z 200 10 157 i- ", 85 %8 8 50 -25 83 27 13 27 10 24 8 22 5 19 3 17 31 14 28 MAR APR MAY JUN JUL AUG FIGURE 1. Singingrates of male and female Northern Cardinals.(Valuesare averagesfor seven-dayperiodsbeginningon the dates indicated.Six femaleswereobserved in 1983, four in 1984, and five in 1985. Five males were observedin 1985.) almost all singing by females occurredbefore nestinghadbegun.Finally,singingfemaleswere usually accompanied by singing mates. The fifteen females under observation sang for a total of 207 min and werejoined by "duetting" mates for 162 min, or 78% of the time. This value increasesto 87%if one exceptionalsinging bout is removed from the analysis. On 20 April 1985 an unbanded female was sighted near the territorialboundaryof the pair under observation. After this female began to sing, the territorialmale flew to a perch near the center of the territoryand also began to sing. Afternearlytwo minutes the unbandedfemale flew acrossthe boundaryand was chased, first by the male and then by the female. Shortly thereafter,the two females began to sing from perchesabout 35 m apart and continued, unaccompaniedby any males, for nearly22 min. PLAYBACKEXPERIMENTS The playbackexperimentsperformedin 1983 revealed that females responded similarly to the songs of males and females (Tables 1 and 2). Typically, females responded to playback of both male and female songs by moving a few meters closer to the speakerand uttering a significantlygreaternumberof chipcalls (P < 0.05). Only two females sang in response to male songs (Table2), and no singingwas noted in responseto female songs (Table 1). In similar experiments with males, all individuals tested (n = 6) respondedto playbackof male and female songs with a significantincreasein number of songs (P < 0.05). The observation of 20 April 1985 noted above appearedto indicate that female song may be importantin certainfemale-femaleinteractions.Tests were conductedin 1985 in an This content downloaded from 157.89.111.52 on Tue, 14 Jan 2014 12:28:19 PM All use subject to JSTOR Terms and Conditions 158 GARY RITCHISON TABLE 1. Responses of females to the songs of neighboringfemales. Closest approach Pre-testperiod (PTP) Test period (P) Post-testperiod (PP) PTP vs. P PTP vs. PP P vs. PP songs Flights 32.5a 0 0 0 1.4 5.9 4.2 3.2 28.9 15.0 NS NS NS 0.05 NS NS 18.1 26.2 - NSb NS NS Chips Valuesfor responsesareaveragesfor all twelvetests. bThe significancelevelsareaccordingto Wilcoxonsigned-ranks tests,twotailed;P < numbergiven;NS = not significant. a attempt to further examine this possibility. During these playback experiments an "intruding"femalecardinal(i.e., a studyskin)was placed near the speaker.During playback,femalestypicallyapproachedto within a few meters of the study skin or even closer. Four of the six females approachedto within 1 m or less and two females made contact with the skin. Femalesalso respondedwith a significant increase in number of chip calls (P < 0.05). Few songswereutteredin responseto playback (Table 3). DISCUSSION My observations,and those of others (Shaver and Roberts 1933, Laskey 1944, Lemon 1968b), indicate that female Northern Cardinals singprimarilyduringthe weeksjust before the initiation of nesting. In contrast,male cardinalsbegin singingmonths before nestingbegins. Typically,males begin to sing and defend territoriesin late Januaryor early February, and most territories are well established by early March(Laskey 1944, Gould 1961, Lemon 1968a). Thus it is not until territorieshave alreadybeen establishedthat females begin to sing. The persistent singing of male passerines during the period of territorialestablishment is well documented (Armstrong 1963, Welty 1982). Such persistence is apparent in male NorthernCardinals,with individuals uttering TABLE2. Responses of females to the songs of neighboringmales.a Closest approach No. Notes/ Pre-testperiod (PTP) Test period (P) Post-testperiod (PP) PTPvs. P PTP vs. PP Pvs. PP 29.7 21.0 18.1 NS NS NS songsb song Flights 0 0.9 1.4 NS NS NS Valuesand significancelevels as in Table 1. bTwo females responded. a Closest approach No. Notes/ No. (m) (m) TABLE 3. Responses of females to the songs of nonneighboringfemalesand a female model.a 6.1 8.5 NS NS NS Chips 1.0 3.7 3.3 12.9 2.0 10.9 NS 0.05 NS NS NS NS (m) Pre-testperiod (PTP) Test period(P) Post-testperiod (PP) PTP vs. P PTP vs. PP Pvs. PP songsb song Flights 23.0 0 7.1 2.3 6.3 12.2 0 0.05 NS NS NS NS NS - Chips 0.5 1.1 5.6 32.6 0.6 12.8 NS 0.05 NS NS NS NS Valuesand significancelevels as in Table 1. bFourfemales responded. a as many as 100 songs per hour (Fig. 1). In contrast, females typically sing fewer than 10 songsperhour(Fig. 1).Suchinfrequentsinging suggeststhat female songis of little importance in the establishmentand maintenanceof territory (Farabaugh1982). The responsesof femaleNorthernCardinals to the playbackof conspecificsongs is also of interest.Althoughfemalesutteredchip calls in response to playback, I noted no significant increase in singing, with or without a female model present.In similartests, male cardinals sang significantlymore songs in response to playback. If singing by females served a territorial function, an increase in singing rates might have been expected during playback. Sucha responsehas been noted in female RedwingedBlackbirds(Beletsky1983)and has also been noted in males in numerousother species (e.g., Weeden and Falls 1959, Goldman 1973, Brooks and Falls 1975, Wunderle 1978, Ritchison 1983b). As noted above, female NorthernCardinals singprimarilyaftermales have establishedterritories and before nesting begins, i.e., during the period when pair-bondsare being formed (Shaverand Roberts 1933, Land 1952, Kinser 1973). Such timing suggeststhat female song may be importantin the establishmentof pair bonds. In addition, I found that females are generallyaccompaniedby singingmates. Lemon (1968b) reportedsimilar observations.He observed a total of 104 min of female song in cardinalsand indicated that females were accompanied by males for 97 min. Farabaugh (1982) suggestedthat such overlappingbouts of songs given by members of a mated pair can be consideredduets. It has been suggested that such duets may play a role in establishing pair bonds, synchronizingreproductiveactivities, or establishingand maintainingterritories (Armstrong 1963, Wickler 1980, Farabaugh 1982). The evidence presentedindicates that singing by female cardinalsplays little or no role in the establishmentand maintenanceof territory. The observation of 20 April 1985 de- This content downloaded from 157.89.111.52 on Tue, 14 Jan 2014 12:28:19 PM All use subject to JSTOR Terms and Conditions SINGING OF FEMALENORTHERN CARDINALS scribed previously indicates that, on rare occasions, female song may be used in interactionswith other females. However, all other evidence, including the timing and relative rarityof female song and the duets with mates, indicates that pair bonding and, perhaps, reproductive synchronization are the most importantfunctionsof singingby female Northern Cardinals. 159 GOULD, P. J. 1961. Territorialrelationshipsbetween Cardinalsand Pyrrhuloxias.Condor63:246-265. G. W. 1973. Ecologyand behaviorof the Cardinal in southernIndiana.Ph.D.diss., IndianaUniv., Bloomington. LAND, H. C. 1952. The seasonal shiftingof behavioral patterns related to territories in the cardinal. M.Sc.thesis,Ohio State Univ., Columbus. LASKEY, A. R. 1944. A study of the cardinalin Tennessee. Wilson Bull. 56:27-44. LEMON, R. E. 1965. The song repertoiresof cardinalsat London, Ontario.Can. J. Zool. 43:550-569. LEMON, R. E. 1967. The responsesof Cardinalsto songs ACKNOWLEDGMENTS of differentdialects.Anim. Behav. 15:538-545. I thankR. E. Lemon, B. M. Gottfried,P. Stettenheim,K. LEMON, R. E. 1968a. The displaysand call notes of CarG. Beal and an anonymous reviewer for many helpful dinals. Can. J. Zool. 46:141-151. comments on the manuscript.Financial assistance was LEMON, R. E. 1968b. The relationbetween the organization and function of song in Cardinals.Behaviour providedby The FrankM. ChapmanMemorialFund of the AmericanMuseumof NaturalHistoryand by Eastern 32:158-178. KentuckyUniversity. NOTTEBOHM,F. 1975. Vocal behaviorin birds, p. 287332. In D. S. Farnerand J. R. King [eds.], Avian biology, Vol. 5. AcademicPress,New York. LITERATURECITED RITCHISON,G. 1983a. The functionof singingin female E. A. 1963. A study of bird song. Oxford Black-headedGrosbeaks (PheucticusmelanocephaARMSTRONG, Univ. Press, London. lus):family-groupmaintenance.Auk 100:105-116. L. D. 1982. Vocalizations of female Northern BELETSKY, RITCHISON,G. 1983b. Responsesof Black-headedGrosOrioles.Condor84:445-447. beaks to songs of conspecifics.Wilson Bull. 95:132L. D. 1983. Aggressive and pair-bond main138. BELETSKY, tenancesongsof femaleRed-wingedBlackbirds(Age- RITCHISON, G. 1984. A new method of markingbirds. N. Am. Bird Bander9:8. laiusphoeniceus).Z. Tierpsychol.62:47-54. BENT, A. C. 1968. Life historiesof North AmericancarSHAVER, J. M., AND M. B. ROBERTS. 1933. A briefstudy of the courtship of the EasternCardinal.J. Tenn. dinals, grosbeaks,buntings, towhees, finches, sparAcad. Sci. 5:116-123. rows, and allies. U.S. Natl. Mus. Bull. No. 237. W.H. BROOKS, R. J., AND J. B. FALLS. 1975. Individual recSOKAL, R. R., ANDR. J. ROHLF. 1969. Biometry. ognitionby song in White-throatedSparrows.I. DisFreeman,San Francisco. criminationof songsof neighborsand strangers.Can. WEEDEN, J. S., AND J. B. FALLS. 1959. DifferentialreJ. Zool. 53:879-888. sponse of male Ovenbirds to recorded songs of neigh- S. M. 1982. The ecological and social sigFARABAUGH, nificanceof duetting,p. 85-124. In D. E. Kroodsma and E. H. Miller [eds.], Acoustic communicationin birds, Vol. 2. AcademicPress, New York. GOLDMAN, P. 1973. Songrecognitionby Field Sparrows. Auk 90:106-113. GOTTFRIED,B. M., AND A. H. GOTTFRIED. 1978. Preliminarystudiesof the vocal responsesof territorialCardinals (Cardinaliscardinalis)to songs of a strange male. Ohio J. Sci. 78:85-87. KINSER, boring and more distant individuals. Auk 76:343- 351. WELTY, J. C. 1982. The life of birds, 3rd Ed. Saunders College Publishers, Philadelphia. WICKLER, W. 1980. Vocal duettingand the pair bond: I. Coyness and partner commitment. A hypothesis. Z. Tierpsychol. 52:201-209. WUNDERLE, J. M., JR. 1978. Differentialresponseof territorial Yellowthroats to the songs of neighbors and non-neighbors.Auk 95:389-395. This content downloaded from 157.89.111.52 on Tue, 14 Jan 2014 12:28:19 PM All use subject to JSTOR Terms and Conditions
Mate Choice in Adult Female Bengalese Finches: Females Express Consistent Preferences for Individual Males and Prefer Female-Directed Song Performances Jeffery L. Dunning, Santosh Pant, Aaron Bass, Zachary Coburn, Jonathan F. Prather* Program in Neuroscience, Department of Zoology and Physiology, University of Wyoming, Laramie, Wyoming, United States of America Abstract In the process of mate selection by female songbirds, male suitors advertise their quality through reproductive displays in which song plays an important role. Females evaluate the quality of each signal and the associated male, and the results of that evaluation guide expression of selective courtship displays. Some studies reveal broad agreement among females in their preferences for specific signal characteristics, indicating that those features are especially salient in female mate choice. Other studies reveal that females differ in their preference for specific characteristics, indicating that in those cases female evaluation of signal quality is influenced by factors other than simply the physical properties of the signal. Thus, both the physical properties of male signals and specific traits of female signal evaluation can impact female mate choice. Here, we characterized the mate preferences of female Bengalese finches. We found that calls and copulation solicitation displays are equally reliable indicators of female preference. In response to songs from an array of males, each female expressed an individual-specific song preference, and those preferences were consistent across tests spanning many months. Across a population of females, songs of some males were more commonly preferred than others, and females preferred femaledirected songs more than undirected songs, suggesting that some song features are broadly attractive. Preferences were indistinguishable for females that did or did not have social experience with the singers, indicating that female preference is strongly directed by song features rather than experiences associated with the singer. Analysis of song properties revealed several candidate parameters that may influence female evaluation. In an initial investigation of those parameters, females could be very selective for one song feature yet not selective for another. Therefore, multiple song parameters are evaluated independently. Together these findings reveal the nature of signal evaluation and mate choice in this species. Citation: Dunning JL, Pant S, Bass A, Coburn Z, Prather JF (2014) Mate Choice in Adult Female Bengalese Finches: Females Express Consistent Preferences for Individual Males and Prefer Female-Directed Song Performances. PLoS ONE 9(2): e89438. doi:10.1371/journal.pone.0089438 Editor: Melissa J. Coleman, Claremont Colleges, United States of America Received July 3, 2013; Accepted January 22, 2014; Published February 18, 2014 Copyright: ! 2014 Dunning et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by startup funds provided by an Institutional Development Award from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P 30 GM 103398. This research was also supported by the University of Wyoming INBRE Program of the National Institutes of Health (JFP) and by an Undergraduate Research Fellowship (ZC) from the University of Wyoming EPSCoR Program of the National Science Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Jonathan.Prather@uwyo.edu reproductive signals and the individual-specific nature of the cognitive behaviors through which those signals are evaluated can have a profound impact on female mate choice. Female songbirds are an excellent model in which to investigate how the features of reproductive signals and the individual-specific features of signal evaluation combine to influence a female bird’s choice of a specific male suitor. In many songbird species, including the Bengalese finches (BFs) studied here, song is used by males to attract female mates, and males sing but females do not [13,14]. Yet despite their inability to sing, female songbirds are equal or superior to males in their ability to perceive the fine details that distinguish the songs of different species or the songs of different males within a species [15,16,17]. In tests of the degree to which female mate preference is influenced by song as opposed to visual characteristics such as beak color, song plays the more important role in the species where that has been tested [18,19]. The importance of song in shaping female mate preference is especially clear in cases when females perform a copulation solicitation display (CSD) in response to song, briefly adopting a posture reminiscent of mammalian lordosis that facilitates male Introduction In the context of mate selection by female songbirds, male suitors seek to attract female mates through their song performances and other signals such as plumage or reproductive displays. Females detect those signals and use that sensory information to form a subjective evaluation of the quality of each signal and the associated male. The results of that cognitive behavior then guide the female’s expression of selective motor responses such as approaching only the preferred male, responding with courtship displays, performing some operant task, or assuming specific postures to initiate copulation [1,2,3,4,5,6]. Some studies reveal that females express broad agreement in their preferences for specific signal characteristics, indicating that those features are especially salient in female mate choice [2,3,7,8,9,10]. Interestingly, other studies reveal that individual females differ in their preference for specific characteristics, indicating that the female’s evaluation can be influenced by individual-specific features of perception rather than simply the physical properties of the signal [11,12]. Thus, both the physical properties of PLOS ONE | www.plosone.org 1 February 2014 | Volume 9 | Issue 2 | e89438 Mate Choice in Female Bengalese Finches In this study, we characterized the song preferences of adult female Bengalese finches. In Phase 1 of this study, we address the hypothesis that female BF mate choice is evident not only in the expression of copulation solicitation displays but also in the number of calls that the female performs during exposure to song stimuli. We predict that if CSDs and calls are equally reliable indicators of BF mate choice [33], then we should observe a strong correlation between each female’s most-preferred song stimulus as revealed by each indicator. In Phase 2, we address the hypothesis that female BF mate preference is directed by the properties of the song stimulus itself and does not require experience with the associated singer. We predict that although the identity of the most preferred male may vary across different females, each female BF’s preference for her most-preferred male will be consistent across time and repeated tests. We further predict that if preference is dictated by the properties of the song signal itself, then preferences will be broadly similar between females that have interacted with the associated male singers and other females that have not interacted with those males. If supported by the data, a finding that female mate preferences are consistent within individuals and do not require specific aspects of past experience with the associated singer (e.g., mate, cage partner, etc.) would vastly facilitate subsequent investigations of the neural circuitry through which songs are perceived and evaluated in this species. Finally, we also address the hypothesis that female BFs prefer directed songs more than undirected song. We predict that female BFs will prefer directed song, and that subtle differences exist between undirected and directed performances by one and the same male (for a similar investigation in zebra finches, see also [30,32]). Such a result would reveal that those subtle differences are functionally significant, and those data could guide subsequent identification of one or more song features that are especially salient in affecting female BF song preference. Thus, the results of this study will reveal the nature of female BF signal evaluation and mate choice. Because we have collected these data in a species in which it is relatively easy to raise individuals under carefully controlled conditions and to collect neurophysiological data during behavioral tests, these insights will set the stage for additional investigation of the developmental processes through which individual-specific preferences emerge and the neural circuits through which sensory signals are evaluated and used to direct mate choice. reproductive access. In fact, song is such a powerful influence on female mate choice that females will perform CSDs in response to songs played through a speaker, even with no male physically present [2,5]. Therefore, CSDs are a well-established and easily interpreted means of assessing female songbird mate choice, and quantifying their expression can be used to identify songs that an individual female finds especially attractive. Features of those songs can be compared against the features of songs that she finds less attractive to identify parameters that may be especially salient in shaping the preferences of individual females. The BFs studied here provide an important advantage in that regard, as the songs of an individual male vary in their note sequence within and across songs [13]. That natural variation makes BF songs more complex than those of other commonly studied species, providing enhanced resolution in efforts to define which song parameters are most salient in shaping female preference. Furthermore, BFs fare especially well and display their full spectrum of courtship and reproductive behaviors in the laboratory. These features make BFs excellent candidates for comprehensive investigation of the mechanisms through which females evaluate the quality of male signals and use that information to guide mate selection. Previous experiments have provided some insight into the nature of female mate choice and the relation between that preference and the features of male song. Specifically, studies of other species have revealed that females can recognize individual males by their songs, and female preferences for the songs of specific males are consistent across time and trials [20]. Comparative approaches have also identified features of male song that are important in directing the preference of females of different species. Female mate choice has been linked to many challenging aspects of song performance including long duration [7,10], loud amplitude [21], combinations of large bandwidth and rapid trilling [3], the inclusion of specific syllables [8,9,22], and the complexity of song content or sequencing [5,23,24,25,26,27]. Thus, studies of other species reveal that female preferences for specific songs and therefore specific males are associated with specific song parameters. In the few studies that have investigated mate selection in BFs, the relation between male song and female preference is much less defined. Some studies have identified the number of different song notes [5] and the complexity of note sequence as contributors to female preference [28]. However other work from the same group reveals little or no relation between note sequence complexity and female preference [25,29]. This lack of clarity leaves open fundamental questions regarding the nature of female BF preference and its relation to the parameters of male song. Specifically, it remains unresolved whether variability in female BF mate preference emerges because song complexity plays little or no role in directing female preference, or whether complexity is influential in some cases but females are highly variable in the nature of the song features that each female prefers. Relevant to that consideration, males change many song parameters when they sing either alone (‘‘undirected’’) or in the presence of a female (‘‘directed’’) [30,31], and those changes are functionally relevant, as females typically prefer songs performed in the directed condition [32]. The distributed nature of changes in the directed condition suggests that one parameter is likely not the sole determinant of female BF preference. Therefore, essential next steps toward understanding the basis of signal evaluation and mate selection in this species are to determine whether song preferences are consistent within individual females, whether song preferences are consistent across individuals, and whether multiple song parameters can be evaluated independently of one another. PLOS ONE | www.plosone.org Methods Ethics Statement We performed all experiments using adult (age . 120 days posthatch) male and female Bengalese finches (BF, Lonchura striata domestica) obtained from our breeding colony or from a commercial breeder. All procedures in this study were approved by the University of Wyoming Animal Care and Use Committee, and procedures were in compliance with recommendations from that group and state and federal regulations governing the housing and use of songbirds. Care and Handling of Experimental Subjects Prior to experimentation, we identified males by their song performance, and we identified females by the presence of calls but the absence of song over three or more days of continuous recording. We housed animals in a colony setting prior to the experimental protocols detailed below, and throughout the experiment we maintained the 15:9 light:dark photoperiod used in our colony. Before beginning behavioral tests, we removed female subjects from the colony and placed them in sound 2 February 2014 | Volume 9 | Issue 2 | e89438 Mate Choice in Female Bengalese Finches stimuli in a random sequence with an interval of 20 to 25 sec (randomly specified by computer) of silence between each song, and we presented each aggregate song once per test. In an attempt to prevent overexposure and habituation to the stimuli, we did not test any bird more than two times in a given day. attenuating chambers in all-female groups of no more than 5 birds for a minimum of 3 days to isolate those birds and prevent them from hearing song or interacting with male birds [26]. During the period when the birds were tested, subjects remained in these isolated groups, but we kept no bird in this condition for more than 14 consecutive days before returning them to the colony for a minimum of 3 days. At the time of each behavioral test, we moved the female subject from this isolation chamber (41 cm631 cm624 cm) to the chamber where we performed our behavioral tests (41 cm633 cm625 cm). We allowed at least 30 minutes to pass between moving the bird into the behavioral testing chamber and the beginning of testing [34]. In addition, we began testing only after the bird’s behavior indicated that it had become comfortable in its new surroundings (e.g., feeding, grooming; tests typically began 30 min after transition). Quantifying Female Responses to Song Stimuli We measured each female’s behavioral responses to presentation of song stimuli by playing song through a speaker (Sound Acoustics) and viewing images and listening to audio captured through a camera (General Electric model 45231) located outside the bird’s cage but inside the sound attenuating chamber. Using these tools, we could clearly see and hear the female subject throughout experimentation. We scored all responses in real time, and we kept a recording of each test on video tape in the event that it was necessary to review the data to ensure accuracy. During song presentation, females responded with many behaviors including the calls and copulation solicitation displays quantified in the Results. A copulation solicitation display is the most easily interpreted indicator of female mate choice. We recognized the occurrence of a CSD when the bird adopted a posture with its head down, its tail feathers slightly raised and often slightly fanned, and its wings either spread out to the side or slightly above the height of the bird’s back [35]. Females typically held that posture for approximately 2 seconds. We detected no clear gradation of CSD expression in our birds’ behavior, therefore we did not attempt to score subtle features of CSD gradation as others have done [35,36]. Because our microphone was close to the bird’s cage, we could detect calls of even relatively low amplitude (e.g., when the bird was not facing the microphone), and we could often visually confirm the occurrence of a call as a brief opening of the bird’s beak. Other behaviors included vigorous perch hopping and occasional beak swiping or wing flaps [26]. Most females exhibited perch hopping at very high frequency across many or all treatment conditions [26]. Females expressed wing flaps less commonly, but they occurred across many or all treatment conditions, and it was often difficult to disambiguate wing flaps from postural adjustments associated with perch hopping. We excluded these measures (perch hopping and wing flaps) from further analysis, and the Results reported here are based on the expression of copulation solicitation displays or calls. Creation and Presentation of Song Stimuli We recorded the songs of 8 individual BF males for at least 24 hours (range 24-36 hours) in a sound attenuation chamber in which we provided seed and water ad libitum. We monitored vocal behavior using a microphone (Shure model SM57) positioned immediately adjacent to the bird’s cage (41 cm631 cm624 cm) and custom software to continually record sounds and save them onto a computer hard drive using custom software (Sound Analysis Pro; songs were bandpass filtered 300 – 10000 Hz; Matlab software). We recorded each male twice in the sound attenuating chamber, once while alone (‘‘undirected’’) and once in the presence of a female (‘‘directed’’). In the behavioral tests described in the Results, we did not use any of the females that were used to evoke directed song, and we were careful to ensure that none of the songs in the directed condition contained female calls or other cage noise. We used recordings of undirected song to represent each of the 8 males in tests of female preference for one among many males. We randomly selected 5 songs performed by each male and screened them only by duration so that the total duration of all 5 songs together was approximately 60 sec (duration of individual songs: 10.1860.36 sec). We concatenated the songs of each male into one aggregate song stimulus containing all 5 songs with 700 ms of silence inserted between each song. None of these individual or concatenated songs had introductory notes, as introductory notes were either absent in the original song performance or we excluded them when individual songs were assembled into the aggregate song stimulus. Thus, we represented each male by 1 aggregate song stimulus containing a string of songs collected in the undirected condition and comprising approximately 60 sec of sound and silence together (duration of aggregate undirected song stimuli: 56.8861.77 sec). We also repeated this process of song selection and assembly using a set of directed songs and another set of undirected songs performed by the same set of 8 males, and we used the resulting set of stimuli to test female responses to directed versus undirected song performances (duration of aggregate undirected song stimuli: 60.6363.77 sec; duration of aggregate directed song stimuli: 61.6363.78 sec; ratio of directed:undirected duration for each male 1.0260.01; durations were indistinguishable in each condition: paired t-test, t(7) = 1.59, p = 0.15). We normalized the amplitude of all song stimuli such that each song had the same standard deviation of amplitude. During experimentation, we housed females individually in a cage inside a sound attenuation chamber (additional details below) and we presented song stimuli at 70 dB through a speaker located 5 to 22 cm away (70 dB measured 13 cm from the speaker, distance from speaker varied according to the bird’s location in the cage). We played song PLOS ONE | www.plosone.org Overview of Behavioral Testing In one set of experiments (Phase 1, Figure 1), we defined the mate preference of each female BF by quantifying the expression of CSDs in response to playback of undirected song from each of 8 different BF males (N = 16 female birds). In those experiments (detailed in the following section), we also quantified the expression of calls during song playback to investigate the degree to which mate preference is evident in not only CSDs but also in expression of other forms of behavior. As detailed in the Results and as has been noted for another songbird species [33], we found that calls are as informative as CSDs in revealing female preference for specific songs. Therefore, we used calls to measure mate preference in all subsequent experiments. In another set of experiments (Phase 2A, Figure 1), we investigated the degree to which female BFs express a preference for the song of one or more individual males and the degree to which that preference is consistent across time and repeated tests (N = 21 female birds that included none of the 16 birds used in tests of CSDs). In a final set of experiments (Phase 2B, Figure 1), we investigated the degree to which each female BF expresses a preference for directed song versus undirected song performed by her most-preferred male (N = 18 female birds that were a subset of 3 February 2014 | Volume 9 | Issue 2 | e89438 Mate Choice in Female Bengalese Finches results of estradiol in BF [5,37] and have used other measures of female BF response to song [28]. We administered 17-ß-estradiol by subcutaneous implant in silastic tubing (1.96 mm outer diameter) containing 8 mm of hormone [6]. During the implantation procedure, the bird was restrained manually, a local anesthetic (4% lidocaine cream) was applied, the implant was inserted through a small incision in the skin overlying the abdomen, and the incision was sealed (VetBond). We monitored the bird throughout recovery. Seven days following that procedure, we presented female BFs with playback of the aggregate undirected song stimuli from the 8 males described above, and we recorded the expression of CSDs to define the mate choice of each female. We tested each bird’s preference 3 times in this paradigm. Our female subjects expressed CSDs following estradiol implantation, but estradiol can affect auditory signal processing [38], and it remains unknown what effect that implant may have on the mechanisms of signal perception and evaluation over long periods. In an attempt to measure female BF mate choice over longitudinal studies in the absence of estradiol, we investigated the degree to which subjects expressed other behaviors in association with the performance of CSDs. We recorded our birds’ expression of calls and CSDs during song playback, and we tested birds in both the control and the hormone-implanted condition. In one set of subjects (N = 6), we tested each bird’s response first in the control condition and then again 7 days following estradiol implantation. In another set of subjects (N = 6, entirely distinct from the 6 birds in the previous group), we tested the birds first in the hormone-implanted condition and then again 6 weeks after removal of the implant to permit recovery from surgery and the effects of the implant. In a third set of birds (N = 4), we tested each bird’s preference following administration of a biologically inert sham implant (Kwik-Sil, World Precision Instruments) of similar size and shape as the hormone implant. Figure 1. Overview of experimental methods and flow. 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. doi:10.1371/journal.pone.0089438.g001 Quantifying Preference for Song of an Individual Male (Phase 2A) In the tests of Phase 2A, we presented females with the aggregate undirected song stimulus for each of 8 males (the same stimuli used in Phase 1), and we recorded responses to quantify each female’s preference for songs of different individuals. We tested each bird’s preference 5 times in this paradigm. Numbers of responses could vary considerably between birds and even between tests of the same bird. To ensure that each test included sufficient data that a preference could be determined, we enforced the criterion that a valid test of preference was one in which the female called at least 10 times in response to the song of one individual male or at least 4 times each to the songs of at least 2 different males. If a bird failed to reach 5 valid tests by the time that it had been tested 10 times, then testing for that bird was discontinued and it was excluded from these results (all birds responded with at least one call to one song stimulus, but 9 birds did not meet this criterion and were excluded). We considered females to be valid if they reached 5 valid tests before that point (21 birds passed). Thus, in Phase 2A we quantified the number of calls that each female produced in response to each male bird and used those data to identify the most-preferred male for each female. Calls produced by our birds were multi-note trills of the sort described previously as amplitude modulated calls in adult female Bengalese finches [39]. The number of trilled notes in each call ranged from 2 to 4, and each bird produced each type of trill at least once in response to the most-preferred song. Our initial analyses revealed no difference between the length of trills (number of trilled notes) that each female produced in response the 21 birds used in Phase 2A). Methods through which preference was computed from the outcomes of tests in Phase 1, Phase 2A and Phase 2B are detailed at the end of this Methods section. Hormone Implantation Surgery and Tests of Copulation Solicitation Displays (Phase 1) In the laboratory, female songbirds generally require a subcutaneous implant of estradiol in order to express CSDs commonly [2,3,6]. That was also the case in the BFs studied here (Figure 2A-2B), however other authors have reported varying PLOS ONE | www.plosone.org 4 February 2014 | Volume 9 | Issue 2 | e89438 Mate Choice in Female Bengalese Finches Figure 2. CSDs and calls are equally reliable measures of female BF mate preference. (A) With an estradiol implant administered, female BFs expressed CSDs in response to song playback, and song identity significantly affected that response. The most-preferred song evoked a significantly greater response than intermediately preferred songs or the least-preferred song (N = 12 birds; for all panels * indicates cases that are different than the response to the most-preferred song (Tukey’s HSD, p , 0.05); statistics are provided in the text unless otherwise noted here). (B) In the absence of an estradiol implant, birds never produced CSDs in response to any song stimulus (N = 12 birds). In no case did a sham implant induce birds to express CSDs in response to song (N = 4 birds, data not shown). (C) Calls produced during song presentation were also tallied in the same tests described in panels A and B. In the presence of an estradiol implant, birds called in response to many songs, and the song that evoked the greatest number of calls was invariably the same song that also evoked the greatest number of CSDs (intermediately-preferred songs and leastpreferred songs evoked responses that were not only significantly different from the response to the most-preferred song (*, Tukey’s HSD, p , 0.05) but also different than one another (dagger symbol, Tukey’s HSD, qs = 3.65, p = 0.04, N = 12 birds). (D) In the absence of an estradiol implant, birds also called in response to song stimuli (intermediately-preferred songs and least-preferred songs evoked responses that were not only different than the response to the most-preferred song (*, Tukey’s HSD, p , 0.05) but also different than one another (dagger symbol, Tukey’s HSD, qs = 4.47, p = 0.01, N = 12 birds). The song that evoked the greatest number of calls without an implant was the same song that evoked the greatest number of calls in the presence of the estradiol implant in 11 of 12 birds. Therefore, calls are an excellent measure of mate preference in adult female Bengalese finches. More calls were produced in the estradiol condition than in the absence of estradiol, but that difference was not significant for any of responses to the most-preferred song (Mann-Whitney U test, p = 0.30, N = 12 birds) or intermediately-preferred songs (p = 0.17) or least-preferred songs (p = 0.91). doi:10.1371/journal.pone.0089438.g002 undirected song stimuli and used that number to calculate a selectivity index for each bird. This index is the magnitude of the female’s response to her most-preferred male divided by the average response across all males. This value is 1 for a female that is equally responsive to all males and much larger for birds that are strongly selective for the song of an individual male(s). to her most-preferred song versus other songs (One-way ANOVA, F(7,72) = 1.54, p = 0.17, N = 10 birds). Furthermore, calls were also commonly accompanied by vigorous activity including approaching the side of the cage nearest the speaker. Because of the dimensions of the testing apparatus, it was difficult to assess the relation between approach and preference, but approaching the source of the sound was commonly observed in association with songs that evoked large numbers of calls. We tallied the total number of calls that each female performed in response to all PLOS ONE | www.plosone.org 5 February 2014 | Volume 9 | Issue 2 | e89438 Mate Choice in Female Bengalese Finches Quantifying Preference for Directed Song (Phase 2B) more ordered structure, with negative infinity representing a completely ordered signal. Number of unique note types in each song: This is the number of distinct note types present in each song. Sequence transition entropy: We computed this value using the methods described in Sakata et al 2008 [31]. A value of 0 indicates that the syllable sequence is completely fixed. Songs containing progressively more variable syllable sequences have higher transition entropies. Sequence linearity, sequence consistency, sequence stereotypy: We computed these values using the methods of Scharff and Nottebohm 1991 [41]. Sequence linearity provides a measure of how song notes are ordered, with a value of 1 for a completely stereotyped order and a positive number approaching zero for a highly variable note sequence. Sequence consistency addresses how often a particular note sequence, the most common note sequence in that song, is performed. A value of 1 indicates a completely consistent song in which all note transitions are part of that most common sequence, and lesser consistency is indicated by a smaller value approaching zero for a highly variable note sequence. Sequence stereotypy is computed using the values of sequence linearity and sequence consistency. Sequence stereotypy provides an easily interpretable summary of sequence features, as its value ranges from 1 for a completely stereotyped, highly ordered song to numbers approaching 0 for songs containing highly variable note sequences. Number of epochs of note repetition in each song, number of note repeats in each epoch of repetition, percent of song duration comprising repetition: We computed these values using custom software (Matlab). They represent the number of epochs in each song when any note was repeated, the number of times that the note was repeated in each of those epochs, and the total number of note repeats in each song divided by the total number of notes in the song, respectively. We used these parameters to compare each female’s most-preferred song versus other songs in the stimulus set and to compare undirected versus directed variants of each male’s song. After a bird had completed Phase 2A, we transitioned the female into Phase 2B. That phase consisted of tests designed to measure whether the bird expressed a preference for directed or undirected variants of the song of her most-preferred male. We presented each female with the aggregate undirected song stimulus (approximately 60 sec) and the aggregate directed song stimulus (approximately 60 sec) of her most preferred male, and we recorded responses to quantify each female’s preference for either of those song variants. We tested each bird’s preference 3 times in this paradigm, and we enforced the same criteria for a valid test that we enforced in Phase 2A. We excluded birds from these results if they failed to reach 3 valid tests by the time that we had tested them 6 times (3 birds excluded from the set of 21 birds that passed Phase 2A). To quantify each female’s selectivity for directed versus undirected song, we tallied the total number of calls that each female performed in presentations of undirected and directed variants of the song of her most-preferred male, and we used that to compute the female’s preference for either variant, which we quantified as the percent response to directed song minus the percent response to undirected song. Using this metric, positive values indicate a greater response directed song, and negative values indicate a greater response to undirected song. Considering the Possible Influence of Each Female’s Experience with the Male Singers Used to Test Female Song Preference In each of the experiments in Phases 2A and 2B, we used two separate cohorts of female birds to begin to examine the role of experience in female BF preference for male song. The first group contained females that were alive at the same time as the males that we used to test female preference (N = 10 birds). To our knowledge, none of the females tested in that group had ever mated with any of the males used to test preference, however because of that contemporary relationship, it is likely that those males and females interacted. To control for that possible influence of experience (e.g., auditory, visual, social), we tested a second group of females that all hatched after all of the male singers were deceased (N = 11 birds; testing for the second group began 14 months after the start of testing for the first group). Therefore, the members of the first and second cohort had grossly similar experiences living in the colony, but the members of the second cohort were naı̈ve to the songs of the males used in this study. All handling and testing procedures were identical between the two groups. Statistical Analyses We used appropriate parametric or nonparametric statistical tests to compare results obtained under different conditions, as reported in association with the results of each comparison. Briefly, in Phase 1 we used a two-way ANOVA to compare the number of responses to each of 8 different song stimuli and in 2 different sequences of estradiol implantation (song was one factor with 8 levels, and testing sequence (either estradiol control, or control estradiol) was another factor with 2 levels). Post hoc tests were used to investigate possible differences in the responses to different song types. As elaborated in the Results, those data clearly indicated that the most-preferred song evoked a different response than other song types. To simplify our comparison of the most-preferred song type versus other degrees of preference, we used the number of CSDs performed by each bird in the estradiolimplanted condition to identify three stimulus categories for each bird: 1) the ‘‘most-preferred’’ song that evoked the greatest number of CSDs in that bird, 2) the ‘‘least-preferred’’ song that evoked the fewest CSDs in that bird, and 3) an ‘‘intermediatelypreferred’’ song type. The methods through which we identified the songs that composed each category are as follows. In the case of the most-preferred song for each bird, that unique distinction was always evident in the behavioral response data, as there was never a tie between the number of CSDs evoked by the 1st and 2nd ranked song types. In the case of the least-preferred song type for each bird, it was common that multiple songs would be associated with expression of zero CSDs. In that case, we randomly selected one of those song types as the least-preferred song type. In the case of the intermediately-preferred song type for each bird, we Quantifying Features of Male Songs We computed the following measures for each of the undirected and directed songs of each of the 8 males we used in tests of female preference (values were computed using custom Matlab software unless otherwise noted). Tempo (notes per sec): We computed tempo by dividing the total number of notes by the total duration of each stimulus. Song duration (sec): This is the duration of the entire song, including all epochs of sound and silence. Maximum frequency (Hz), mean frequency (Hz), minimum frequency (Hz), spectral entropy: We computed these parameters using Sound Analysis Pro software [40]. The frequency values were the maximum, minimum, and mean of all sounds present in the complete song stimulus. Spectral entropy (Weiner entropy) is a measure of the width and uniformity of the power spectrum, and we computed this value using algorithms detailed in Sound Analysis Pro and in publications describing that software [40]. Entropy ranges from 0 for complete disorder to progressively more negative values for progressively PLOS ONE | www.plosone.org 6 February 2014 | Volume 9 | Issue 2 | e89438 Mate Choice in Female Bengalese Finches (Tukey’s HSD, qs . 11.44 and p , 0.05 for both comparisons, N = 12 birds). Therefore, the most-preferred song is easily identified by observing CSDs, and next we investigated whether that preference was also evident in the expression of calls. We recorded our estradiol-implanted birds’ expression of calls during song playback and found that song identity also significantly influenced calling behavior (Two-way ANOVA, F(7,95) = 39.53, p , 0.001, N = 12 birds) but the sequence of testing had no effect (F(1,95) = 2.48, p = 0.12, N = 12 birds) and there was no significant interaction (F(1,7) = 1.10, p = 0.37, N = 12 birds). As in the case of CSDs, that influence on calls was also evident even when we categorized our stimuli. Specifically, song identity influenced calling behavior (Figure 2C, Two-way ANOVA, F(2,35) = 51.83, p , 0.001, N = 12 birds), the sequence of testing had no effect (F(1,35) = 0.84, p = 0.37, N = 12 birds), there was no significant interaction (F(1,2) = 1.99 p = 0.15, N = 12 birds), and females called significantly more in response to the most-preferred song than in response to any other stimulus (Figure 2C, Tukey’s HSD, qs . 3.65 and p , 0.05 for both comparisons of most-preferred song type versus other song types, N = 12 birds). When the same birds were tested in the absence of an estradiol implant, the birds did not express CSDs (Figure 2B), but song identity had a significant effect on calling (Figure 2D, Two-way ANOVA, F(2,35) = 76.45 p , 0.001, N = 12 birds), the sequence of testing had no effect (F(1,35) = 2.29, p = 0.14, N = 12 birds), and there was no significant interaction (F(1,2) = 2.92, p = 0.07, N = 12 birds). Even without an estradiol implant, females called significantly more in response to the most-preferred song than in response to any other stimulus (Figure 2D, Tukey’s HSD, qs . 4.47 and p , 0.05 for both comparisons of most-preferred song type versus other song types, N = 12 birds). Therefore, the mostpreferred song is also easily identified by counting the number of calls that the female produces during song playback. To further investigate the relation between CSDs and calls, we compared the percentage of each bird’s total number of CSDs versus the percentage of each bird’s total number of calls that were evoked by each of the most-preferred, least-preferred and intermediatelypreferred song types, and we found good agreement between these measures (Figure 3, Spearman rank correlation coefficient, rs = 0.82, p , 0.01, N = 12 birds). Together, these data establish a link between mate choice and the expression of calls in response to song presentation. In the remaining experiments, we used calls to quantify each female bird’s mate preference. randomly selected a song type from among the remaining 6 stimuli that were neither the most-preferred nor the least-preferred song type. These stimulus categories, which were defined using CSD data in the estradiol-implanted condition, were maintained when we considered the number of calls that each bird produced in response to each song. As detailed in the Results, the song that evoked the greatest number of CSDs invariably also evoked the greatest number of calls. Therefore, the term ‘‘most-preferred song’’ refers to the song that evoked the most CSDs in each bird, and that term also refers to the song that evoked the greatest number of call responses in that same bird. In Phase 2A, we used a one-way ANOVA and post hoc tests to compare the number of calls that each bird produced in response to songs of 8 different males. In Phase 2B, we used chi-squared tests to compare the number of calls that those same birds produced in response to directed and undirected variants of the song of each female’s most-preferred male. In tests of the degree to which song preference is related to experience with the associated singers, we used a Kolmogorov-Smirnov 2-sample test to compare the results obtained from females that were alive at the same time as the males whose songs were used to test preference versus results obtained from birds that were not alive at that time. Finally, we used a one-way ANOVA and post hoc tests to compare the properties of each of the 8 song stimuli used in Phase 2A and each of the directed versus undirected song variants used in Phase 2B (separate tests for each song property). We used the Mann Whitney U test in other comparisons of two quantities in which the data are not parametrically distributed. We express each of our results as mean 6 SE unless otherwise noted. Results Calls during song presentation are an indicator of female sexual preference In Phase 1, we presented female BFs with playback of song from 8 different BF males and counted the number of copulation solicitation displays performed in response to each song following a subcutaneous implant of estradiol (N = 12 birds). For each bird, there was a ‘‘most-preferred song’’ for which more CSDs were performed than in response to the song of any other bird, but many songs evoked no CSDs at all (identity of the most-preferred song varied across females, considered again in later sections). Song had a significant effect on CSD expression in the estradiolimplanted condition (Two-way ANOVA, F(7,95) = 53.51, p , 0.001, N = 12 birds), but the sequence of testing (control estradiol or estradiol implant removed) had no effect on CSD expression (F(1,95) = 0.63, p = 0.43, N = 12 birds) and there was no significant interaction (F(1,7) = 0.31, p = 0.95, N = 12 birds). Females expressed significantly more CSDs in response to the most-preferred song than in response to any other stimulus (Tukey’s HSD, qs . 18.01 and p , 0.05 for all 7 comparisons of most-preferred song type versus all other song types, N = 12 birds). To simplify our comparison of responses to the most-preferred song type versus responses to stimuli with lesser subjective value, we also identified the least-preferred song type and an intermediately preferred song type for each bird (details in Methods). As in the previous result, analysis using this categorization of stimuli revealed that song had a significant effect on CSD expression (Figure 2A, Two-way ANOVA, F(2,35) = 52.12, p = , 0.001, N = 12 birds), the sequence of testing had no effect (F(1,35) = 0.30, p = 0.59, N = 12 birds), there was no significant interaction (F(1,2) = 0.30, p = 0.74, N = 12 birds), and females expressed significantly more CSDs in response to the most-preferred song than in response to the least-preferred or intermediately-preferred stimuli PLOS ONE | www.plosone.org Mate preferences of individual females are consistent across time and tests To characterize mate preference for each bird, we tested each female’s response to a suite of songs from each of 8 BF males. In this Phase 2A of testing, the preference of each female for each of the 8 males was quantified over the course of 5 tests for each bird (e.g., Figure 4A). The pattern of song preference that characterized each female was stable across time and tests spanning intervals as short as 3 hours and as long as 130 days (interval between tests: 13.2262.79 days, N = 21 birds). For example, the bird described in Figure 4A expressed a rather selective preference for the songs of males B and F, and the bird described in Figure 4B was broadly responsive to the songs of many males. Although the identity of the most-preferred male differed between individual females (detailed below), each bird’s preference was consistent across time and tests, as evident in the small standard error bars in Figures 4A-4B and 4D-4E (across all 10 birds standard errors were also quite small: 3.060.4% on the scale used in Figures 4A-4B and 4D-4E, corresponding to a coefficient of variation of 0.3560.03). Consistency in the preference of each individual female indicates 7 February 2014 | Volume 9 | Issue 2 | e89438 Mate Choice in Female Bengalese Finches had encountered those males in the colony, identified specific males by their song, but evaluated the quality of those potential suitors based on other traits such as plumage or social hierarchy. To account for that possibility, we repeated the Phase 2A tests described in Figures 4A-4C, however in this second iteration we performed our tests using adult female BFs that had never interacted with any of the males from which we recorded songs used to test mate preference (‘‘inexperienced group’’, N = 11 birds, Figures 4D-4F). Therefore, there was no possibility that those inexperienced females had any sensory or social experience with the males used in tests of song preference. As in the case of experienced females, these tests using inexperienced females revealed that some females were selective, with strong responses to song of only one or two males (e.g., Figure 4D, selectivity index = 3.76), and other inexperienced females were much less selective (e.g., Figure 4E, selectivity index = 1.60). Preferences were generally very similar between experienced and inexperienced females. Specifically, no difference in selectivity index could be detected between experienced females (Figures 4A-4C) and inexperienced females (Figures 4D-4F, Mann-Whitney U test, p = 0.61; a power test reveals that 81 birds would have been necessary to have statistical power of 0.8). Although this does not exclude the possibility that a subtle difference may exist in the selectivity of these two populations, the general similarity of these values suggests that experience with the singers of specific song stimuli has little or no effect on a female’s selectivity for those stimuli. Individual-specific mate preferences were also consistent for inexperienced females (standard error bars as shown in Figures 4A-4B and 4D-4E were of magnitude 5.0 + 0.4%, corresponding to a coefficient of variation of 0.4260.03). Other features of female mate preference were also similar between the experienced and the inexperienced groups, as described in the following text and figures in which different symbols or different panels are used to distinguish between females with or without experience of the associated male singers. Variability in identity of the most-preferred male. Across the population of females tested here, song identity had a strong influence on female mate choice (Figure 5A, One-way ANOVA, F(7,168) = 80.86, p , 0.001, N = 21 birds). There was also considerable variability in the identity of the male that each female identified as her most-preferred male. Specifically, across the 21 females tested here, there were 5 songs that were ranked as at least one female’s most-preferred song (Figure 5A). To more broadly describe the preferences detected across the population, we also identified not only each female’s most-preferred song (first ranked in Figures 4C or 4F, summarized in Figure 5A) but also the song that each female identified as second most-preferred. When the dataset was analyzed with that more inclusive perspective, we identified 6 songs that were ranked as either most-preferred or second most-preferred by at least one of the 21 females. Specifically, the males designated B and F in Figure 4 were very commonly preferred, with 6 of the 21 females (29%) ranking one of those males as their most-preferred song (Figure 5B). Importantly, the tendency for females to prefer the songs of those two males was evident both in the population that had experience with the associated male singers and the population that had no experience with those males. This broad similarity in the mate preferences of these two groups (Figure 5B; distributions were indistinguishable between groups, Kolmogorov-Smirnov two-sample comparison, p = 0.93, N = 8 songs) reveals that female preference for the song of a specific male does not require sensory or social experience with that male. Instead, female preference is strongly directed by acoustic features of the song itself. Therefore, our data reveal that BF females express a range of individual- Figure 3. Calls are an index of female BF mate choice. 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 (xaxis) 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. doi:10.1371/journal.pone.0089438.g003 that female BFs are capable of recognizing conspecific males by their songs, and female preferences are systematically related to some feature or suite of features that characterize each song. Mate preferences vary across different females Variability in selectivity index. Some female BFs were quite selective in their mate preference, as revealed by strong responses to the song of only one or two males and little or no response to the songs of other males (e.g., Figure 4A, selectivity index = 3.76). Other females were much less selective in their mate preference, as revealed by similar responses to the songs of many or all males (e.g., Figure 4B, selectivity index = 1.43). Together with the consistency of individual preferences across time and tests (detailed above), these differences between birds indicate that each female BF expresses a characteristic pattern of mate preference, with each female typically expressing a strong preference for the song of only one or two males (all 10 birds in this dataset are summarized in Figure 4C, mean selectivity index = 2.6560.31). Mate preference is directed by song rather than interaction with the associated singer. The finding that individual females typically prefer the song of only one or two males suggests that female BF mate preference is related to some acoustic feature or set of features that distinguish those songs. However, another possibility is that females use the song to identify their male of choice [35], but their preference is related to some other aspect of experience with that male. For example, the females that were tested in the dataset described in Figures 4A-4C were all alive at the same time as the males from which we recorded the songs used to test mate preference (‘‘experienced group’’, N = 10 birds). Therefore, it is possible that those females PLOS ONE | www.plosone.org 8 February 2014 | Volume 9 | Issue 2 | e89438 Mate Choice in Female Bengalese Finches Figure 4. Individual females vary in their selectivity for songs of different males. 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 6 SE = 2.6560.31, 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 6 SE = 2.4760.17) 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). doi:10.1371/journal.pone.0089438.g004 PLOS ONE | www.plosone.org 9 February 2014 | Volume 9 | Issue 2 | e89438 Mate Choice in Female Bengalese Finches specific preferences for specific song features, yet there are nonetheless some songs that contain features of sufficiently broadly relevant attractiveness that they evoke at least some courtship behavior from a range of different females. To begin to investigate what features may be most important in distinguishing those broadly attractive songs from the songs of other less attractive males, we quantified a spectrum of properties for the undirected songs of each of the 8 males used in Phase 2A tests of female preference. We found that for only 6 of the 14 properties that we measured, the song of either male B or male F was superlative (had either the greatest of the least magnitude) among the songs of all 8 males. Those 6 properties included mean tempo, mean frequency, spectral entropy, the number of epochs of note repetition, the number of repeated notes in each epoch, and the percent of song duration that comprised note repetition. We further investigated that relation by computing the correlation between the magnitude of those values for each song and the percent of all calls that each song evoked. In only 3 cases was that correlation coefficient (Spearman rs) significant (p , 0.05) and greater than 0.50 (mean tempo, mean number of repeated notes, percent of song duration comprising note repetition). These correlations suggest that song tempo, the extent of repeated notes in the song, or some combination of those features may be important in female evaluation of mate quality. Defining the role of these features together or alone in shaping female preference will require additional experimentation in future studies. Variability in preference for female-directed song. Previous studies of other songbird species indicate that females prefer songs that males perform in the company of a female and direct specifically to that female receiver (‘‘directed’’ songs) more than songs that males perform when they are alone (‘‘undirected’’ songs) [32]. To investigate that possibility for the BFs studied here, we performed Phase 2B tests of each female’s preference for directed versus undirected songs performed by her most-preferred male. Some females expressed a significant preference for directed song (filled symbols in Figure 6, p , 0.05 in chi-squared tests), other birds expressed no significant preference (open symbols in Figure 6), but no female expressed a significant preference for undirected song over directed song. Thus, among those birds that did express a significant preference, all of them preferred directed song over undirected song (N = 18 birds). That tendency was evident both in the group of experienced females (Figure 6, birds 1-10) and in inexperienced females (Figure 6, birds 11-18). Consistent with results from other species [32], our behavioral data reveal that the properties of directed songs are sufficiently different from those of undirected songs that females can recognize them as distinct. Our data also reveal that the differences between directed and undirected songs are relevant to signal evaluation and mate selection. In many cases, those changes cause the song to become more attractive to the female receiver. In other cases, there is little or no effect on the female’s response, however in no case do those changes cause the song to become less attractive than the song performed when the male is alone. Following on our investigation of the properties that distinguished the songs of specific males in Phase 2A, we also quantified a spectrum of properties for the undirected and directed songs of each of those males (Table 1; undirected and directed songs were analyzed from each of the 8 male birds that were the source of the song stimuli used in Phases 1 and 2A of testing). In no case were any of the properties consistently increased or decreased in the directed songs of each male. An especially informative case may be the song of the male that was most broadly preferred among all the females tested and was therefore used most commonly in tests of preference for directed versus undirected song (male B in Figures 4 and 5). For that male, the largest changes between the undirected and directed states (changes of at least 10% of the value in the undirected state) were evident in mean tempo, sequence linearity, sequence stereotypy, the number of epochs of note repetition, and the percent of song duration comprising note repetition (Table 1). Taken together, these changes reveal that the directed song of that male had a faster tempo, was more variable in its sequence, and Figure 5. Females prefer songs of some males more than others. 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 secondranked 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). doi:10.1371/journal.pone.0089438.g005 PLOS ONE | www.plosone.org 10 February 2014 | Volume 9 | Issue 2 | e89438 Mate Choice in Female Bengalese Finches Figure 6. Females commonly prefer directed songs more than undirected songs. 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 Figure 4A, triangle indicates bird in Figure 4B). 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 Figure 4D, diamond indicates bird in Figure 4E). 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. doi:10.1371/journal.pone.0089438.g006 identity and selectivity for directed versus undirected song was evident in birds that had experience with the male singers (Linear regression slope not different than zero: p = 0.72, Spearman correlation not significant, p = 0.67, N = 10 birds, filled symbols in Figure 7) and in birds that had no experience with the male singers (Linear regression slope not different than zero: p = 0.21, Spearman correlation not significant, p = 0.35, N = 8 birds, open symbols in Figure 7). These data reveal that selectivity for one facet of male song does not obligate a female to be equally selective for other facets of that song. Therefore, preference is not a monolithic entity. Instead, female BF mate choice is influenced by multiple facets of male song performance, and at least two of those facets are evaluated independently. Evidence of independent evaluation of specific song features gives rise to the very interesting possibility that evaluation of different song features may be independent cognitive processes associated with activation of distinct populations of brain cells. contained less note repetition than the undirected song of the same bird. These findings leave open the important question of what song features are most salient in directing female mate preference, but they highlight several candidate features and the degree to which they may be positively or negatively correlated with female preference. Preferences for different aspects of male song are independent To begin to understand how perception of song quality may be encoded in the female brain, we investigated the degree to which specific song features are evaluated independently. In other words, we investigated whether a female that was choosy for the identity of an individual singer was similarly choosy for directed versus undirected song. Similar degrees of choosiness for multiple song features would suggest that perception of those different features is accomplished through activation of one and the same neuronal population or separate populations that are strongly linked. In contrast, no relation between the degree of choosiness for different features would suggest that perception of those features is accomplished through activation of separate populations of auditory processing neurons. Thus, an analysis of the degree to which different facets of female preference are correlated with one another will enable us to formulate hypotheses about the neural circuitry through which song-based mate preference emerges. We quantified each female’s selectivity for the songs of different males (selectivity index, Figure 4) and compared that to her selectivity for directed song (Figure 6). Across the population of female birds, there was no significant relation between those variables (Figure 7), evident as a slope that was not different than zero (Linear regression, p = 0.27, regression = 18 birds) and no significant correlation (Spearman correlation, p = 0.28, N = 18 birds). The absence of a relation between selectivity for individual PLOS ONE | www.plosone.org Discussion Female Bengalese finches select their mates based at least primarily on properties of the songs performed by male suitors. The mate preferences of individual females are stable across intervals ranging from hours to months, and establishing a preference for one song but not another does not require that the female has ever had any interaction with the associated male singers. These data do not exclude a role for more general experience in shaping the features of mate preference, however they make clear the importance of the signal in communicating not only the identity of the singer but also the subjective quality of that signal and its sender. Finally, we also found that individual birds could be selective for some song features but not selective for others. The absence of a relation between selectivity for different 11 February 2014 | Volume 9 | Issue 2 | e89438 Mate Choice in Female Bengalese Finches Table 1. Parameters of Directed and Undirected Songs of Each Male Used in these Experiments Male Identity Male A Male C Male D Male G Male H UNDIR. DIR. UNDIR. DIR. Male B UNDIR. DIR. UNDIR. DIR. UNDIR. DIR. UNDIR. DIR. UNDIR. DIR. UNDIR. DIR. Tempo (notes/s) 8.74 8.42 12.93 15.48 8.61 7.70 9.52 8.79 7.15 8.76 9.18 10.99 7.86 11.00 6.66 Song duration (s) 74 77 56 58 77 75 55 54 56 54 59 60 46 47 65 Max. frequency (Hz) 10248 8870 9042 8784 9732 10248 10076 978 Male E 8956 12660 9560 10680 10852 10852 68 10680 806 806 892 892 892 892 806 1150 1064 806 806 822 1064 Mean frequency (Hz) 3384 3472 4138 4263 3800 3436 3836 3934 3884 3788 3793 3678 3618 4085 3909 3843 N different note types 10 10 7 6 6 6 6 7 7 9 8 6 6 Spectral entropy -2.61 -3.06 -2.87 -3.11 -2.53 -2.66 -2.35 -2.51 -2.18 -2.04 -2.95 -2.87 -2.74 -2.16 -2.02 -2.05 Sequence entropy 4.41 4.13 2.85 2.93 3.88 3.68 2.91 2.33 2.34 2.88 3.47 3.64 4.33 4.01 2.58 2.79 Sequence linearity 0.56 0.58 0.47 0.30 0.42 0.27 0.54 0.41 0.50 0.30 0.37 0.25 0.50 0.24 0.49 0.35 Sequence consistency 0.74 0.76 0.74 0.71 0.63 0.58 0.70 0.75 0.70 0.65 0.59 0.56 0.80 0.59 0.74 0.72 Sequence stereotypy 0.65 0.67 0.60 0.51 0.52 0.43 0.62 0.58 0.60 0.47 0.48 0.40 0.65 0.41 0.62 0.54 Number of epochs of note repetition 12 12 12 10 6 7 20 13 8 6 23 16 3 3 7 7 Average number of repeated notes in each epoch 2.38 2.35 3.50 3.49 1.00 1.15 1.41 1.40 1.44 1.30 1.84 1.76 1.25 1.06 1.75 2.03 Percent of song duration comprising note repetition 35.0 28.4 41.5 35.6 14.4 13.8 51.7 29.5 17.3 10.2 51.9 47.4 8.4 9.3 18.7 19.8 7 906 10594 11024 6.41 Min. frequency (Hz) 7 978 Male F 7 doi:10.1371/journal.pone.0089438.t001 facets of male song suggests that evaluation of different parameters occurs independently. Our data forge a link between Bengalese finch sexual preference, evident in a copulation solicitation display, and expression of other overt female behaviors, such as calling in response to hearing a song that the female finds attractive. In some cases, behavioral measures such as hopping onto a perch to evoke song playback (for example, [4]) are used to measure female preference, however those behaviors can leave uncertainty about whether females are engaging with those stimuli because of familiarity or because of a sexual preference for those singers. Other behaviors, such as egg laying [23] or collecting nest materials [28], have also been used as measures of mate preference (for Bengalese finches, see [28]), and the relevance of these behaviors to reproduction is certainly more evident than the relevance of hopping onto a perch. However, the expression of a copulation solicitation display leaves no doubt about the functional relevance of that behavior [6]. Our findings reveal that the song that evoked the greatest number of CSDs also invariably evoked the greatest number of calls, and there was generally good agreement between the number of calls and the number of CSDs associated with the intermediately-preferred and least-preferred song types. Although female songbird mate choice has been studied in a number of different species (reviewed in [36,42]), it has been quite challenging to identify behavioral indices of mate choice that do not require hormonal manipulation (for example [43]) or experimental settings much larger than those commonly found in the laboratory (for example [44]). Our identification of calls as an index of female BF mate choice (for canaries, see [33]) will enable us to assay female mate preference without the invasiveness or other potential complications associated with estradiol implants. Calls have the additional advantages that there is no need to wait for the onset of hormone effects, there is no concern regarding possible changes due to prolonged hormone exposure, and calls are nearly ubiquitous among subjects as opposed to the relative rarity of CSDs [33,45,46]. Together with these benefits, the findings in the present study will enable us to use Figure 7. Different facets of male song can be evaluated independently. 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 Figure 4A, triangle indicates bird in Figure 4B) and females that had no experience with the male singers (open symbols; star indicates birds in Figure 4D, diamond indicates bird in Figure 4E; statistics detailed in the text). Thus, an individual bird can be selective for one aspect of male song but unselective for another aspect. doi:10.1371/journal.pone.0089438.g007 PLOS ONE | www.plosone.org 12 February 2014 | Volume 9 | Issue 2 | e89438 Mate Choice in Female Bengalese Finches calls to probe the properties of female BF mate choice and the features of male song that are salient in that evaluation. Individual female BFs were not identical in their preferences, with many different females identifying different males as their most-preferred suitor. Such individual-specific preferences suggest that rather than being dictated by one dominant song feature, female mate choice is influenced by a suite of features that together define the subjective quality of each stimulus [47]. On the other hand, across the population of birds that we tested there was a trend for 2 males to be more commonly preferred than the other 6 males. The broadly attractive nature of those songs leaves open the possibility that some song features are more influential than others in affecting a female bird’s perception of subjective quality. Thus, the preference of each female reflects both individual-specific features and trends that are broadly shared across the population. Our data reveal that females commonly preferred songs that were performed in the context of a female receiver, and the song changes that occur in that context highlight parameters that may be especially salient in female mate choice. Future experiments to define the relation between changes in those properties and changes in the preference of individual females will provide insight into the forces that have shaped mate selection and the evolution of song characteristics in this species [48]. A particularly intriguing aspect of our dataset is that birds that had not interacted with the males used in these tests report preferences that are very similar to those of females that had interacted with those males. From these data, we can conclude that the subjective value assigned to each song must reflect the quality of the stimulus itself rather than the value of an outcome predicted from previous experience with that singer. Therefore, interaction with a specific male is not necessary for the female to find his song attractive, however this by no means rules out other forms of experience as important factors influencing female mate choice. Studies in other species have revealed that female mate choice is shaped at least partially through experience during a period of early development [49,50], and an important goal of future experiments will be to define how preferences emerge in the development of individual females. The specificity of the preferences detected here suggests an important role for learning in shaping the detailed characteristics of individual preferences [51,52,53,54,55]. Because our birds were kept in group-housed cages where no rearing of offspring occurred, females were not able to learn by observing the outcome of mating. Therefore, stimulus value may have been learned by observing the stimulus preferences of other females. Female songbirds that live in large colonial assemblies can recognize individuals based on their calls [56], enabling eavesdroppers to attend to the preferences of specific individuals and suggesting a mechanism through which birds may detect the preferences of other females and use that information to modify their own preferences. An abundance of calling in some cases and a paucity in others, as we observed in our data, may provide a graded measure of preference that is more nuanced than simply the presence or absence of a CSD. The ease of rearing young BF in carefully controlled social contexts opens the door to fascinating investigations of the degree to which female mate preference may be influenced by social interaction with other females (e.g., to observe their preferences) or other males (e.g., to learn a spectrum of performance ability). Our data also reveal that choosiness for one song facet does not obligate the bird to be choosy for all facets, revealing that multiple song features are evaluated independently. This independence suggests that the mechanisms underlying those aspects of evaluation may include separate populations of neurons, and perhaps even separate brain sites, which participate in detection and evaluation of different facets of song. Specific sites within the auditory forebrain of female songbirds have been implicated in recognition of mate signals [32,57,58,59], and an important future goal will be to couple the behavioral methods used here with highresolution neurophysiological records to define how preference is encoded in the brain. In such studies, a longitudinal approach will be necessary to define the preferences of each individual, making the consistent preferences of individual BF females an especially valuable trait. In our experience, BFs are the easiest species from which to collect neurophysiological data [60]. Female BFs will provide the additional advantage that individuals vary in the identity of the song they prefer most, and that variation will facilitate disambiguation of how the brain encodes subjective value per se as opposed to the physical properties of the stimulus. 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Undirected (Solitary) Birdsong in Female and Male BlueCapped Cordon-Bleus (Uraeginthus cyanocephalus) and Its Endocrine Correlates Nicole Geberzahn*¤, Manfred Gahr Department of Behavioural Neurobiology, Max Planck Institute for Ornithology, Seewiesen, Germany Abstract Background: Birdsong is a popular model system in research areas such as vocal communication, neuroethology or neuroendocrinology of behaviour. As most research has been conducted on species with male-only song production, the hormone-dependency of male song is well established. However, female singing and its mechanisms are poorly understood. Methodology/Principal Findings: We characterised the song and its endocrine correlates of blue-capped cordon-bleus (Uraeginthus cyanocephalus), a species in which both sexes sing. Like other estrildids, they produce directed song during courtship and undirected (or solitary) song in isolation, i.e. when the mate is not visible or absent. We compare solitary song of blue-capped cordon-bleus to published descriptions of the song of its relative, the zebra finch (Taeniopygia guttata). Solitary song of cordon-bleus shared some overall song features with that of zebra finches but differed in spectro-temporal song features, sequential stereotypy and sequential organisation. The song of cordon-bleus was dimorphic with respect to the larger size of syllable repertoires, the higher song duration and the lower variability of pitch goodness (measuring the pureness of harmonic sounds) in males. However, in both sexes the overall plasma testosterone concentrations were low (ca. 300 pg/ml) and did not correlate with the sexually dimorphic song motor pattern. Despite such low concentrations, the increase in the rate of solitary song coincided with an increase in the level of testosterone. Furthermore, the latency to start singing after the separation from the mate was related to hormone levels. Conclusions/Significance: Our findings suggest that the occurrence of solitary song but not its motor pattern might be under the control of testosterone in female and male cordon-bleus. Citation: Geberzahn N, Gahr M (2011) Undirected (Solitary) Birdsong in Female and Male Blue-Capped Cordon-Bleus (Uraeginthus cyanocephalus) and Its Endocrine Correlates. PLoS ONE 6(10): e26485. doi:10.1371/journal.pone.0026485 Editor: David Reby, University of Sussex, United Kingdom Received January 9, 2011; Accepted September 28, 2011; Published October 19, 2011 Copyright: ! 2011 Geberzahn, Gahr. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The study was financed by the Max Planck Society. The European Research Network in Neurosciences CNRS-MPG (GDRE CNRS - MPG Neurosciences) funded NG while working on the manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: nicole.geberzahn@u-psud.fr ¤ Current address: CNPS, CNRS-UMR 8195, Université Paris Sud, Orsay, France Blue-capped cordon-bleus are small, moderately gregarious, sexually dimorphic with respect to plumage colouration and socially monogamous estrildid finches occurring in semi-arid and arid regions of East Africa. They are opportunistic breeders and only defend an area around the nest but no territories [14]. Both sexes sing and their song plays an important role in inter-sexual communication, i.e., song is an advertising signal addressed to mates, whereas it has no function in territorial defence [15]. Like other estrildids, blue-capped cordon-bleus mainly produce song in two different social contexts: (1) during and as part of the courtship display (directed or courtship song) and (2) apart from the courtship context (undirected song), particularly when the mate is not visible or absent. Directed and undirected song has been intensively studied in the zebra finch, a species in which only males sing. Whilst zebra finches use the same song type in both contexts there are subtle differences in the way that song type is performed: song performance is less variable in directed than in undirected song, Introduction Birdsong has been studied mainly in species that breed in the temperate zone, and in most of them, only male birds sing. However, the majority of bird species breed in the tropics [1], and females of many of the tropical species sing as well [2]. The neglected issue of female song production started to attract more attention recently, (e.g. [3–13]), but the physiology (e.g. endocrine control) of female song production as well as of sexspecific differences in singing behaviour are still poorly understood. We have recently established a breeding colony of blue-capped cordon-bleus (Uraeginthus cyanocephalus), to study song production, song development, song learning and endocrine effects on song in this species both in females and males in the laboratory. Blue-capped cordon-bleus are members of the same songbird family (the estrildid finches) as the widely studied zebra finch (Taeniopygia guttata) but differ from the latter in that the females regularly sing. PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e26485 Female Birdsong and Its Endocrine Correlates Methods during directed singing song units are repeated more often, song is performed faster and has a more ‘rigid’ syntax [16]. Furthermore, syllables are produced with significantly higher spectral stereotypy [17]. Such behavioural differences are paralleled by differences in the neuronal activity [18,19]. Cordon-bleus (i.e. species of the genus Uraeginthus) rarely produce undirected song in the presence of the mate, but rather when they are separated from the mate, and therefore their undirected song has also been called solitary song [20,21]. Thus, in cordon-bleus, solitary song is easily amenable to bioacoustic research as it can be reliably induced under controlled conditions by separating members of a pair from each other. In many passerine species the song of females is less complex and/or shorter than in males as for example in red-cheeked cordon-bleus (Uraeginthus bengalus), a congener of our study species [20] or in black-bellied wrens (Thryothorus fasciatoventris) [22]. Song structure can also be just slightly different between females and males as in European starlings (Sturnus vulgaris [23], but see [24]) or indistinguishable between sexes, as for instance in the forest weaver (Ploceus bicolor [25]). The song of passerine birds is generally believed to be under the control of gonadal hormones affecting song control nuclei in the brain by binding to hormone receptors (e.g. [26]). In zebra finches, castration of adult males reduced the amount of both directed and undirected song [27,28]. Although both kinds of song can be restored with testosterone replacement, hormone sensitivity varies; undirected singing has a lower threshold for hormonal activation than directed song [27]. Furthermore, the conversion of androgens to estrogens plays an important role for directed song - but not for undirected song, as inhibiting the transformation of androgen to oestrogen (by aromatase blocker) reduces the likelihood that males produce directed song, while undirected song remains unaffected [29]. The endocrine control of female song is not well understood. Studies on European starlings [30,31] and European robins (Erithacus rubecula, [32]) suggest that like in males, testosterone plays an important role for song production in females. In addition, in many species in which females do not normally produce song or sing rarely, they can be induced to sing by treating them with testosterone, (e.g. [30,31,33–37]). In the zebra finch, however, adult females cannot be induced to sing by testosterone treatment unless they received testosterone or estradiol treatment as hatchlings. Thus, early estradiol action seems to masculinise the female song system and enables the induction of singing behaviour by testosterone administration (e.g. [38–40]). Females masculinised as such may even sing spontaneously without further treatment when adult [41]. Solitary (or undirected) song has never been compared between females and males of a species in which both sexes regularly sing. Also, it is unclear whether the occurrence of solitary song in such a species correlates with an increase in testosterone in both sexes. To answer these questions, we induced members of a pair of blue-capped cordon-bleus to produce solitary song by separating them from each other and took blood samples to determine plasma testosterone levels. We compared the solitary song of females and males by conducting a detailed multi-parametric analysis of their song. Our aims were to (1) describe the solitary song in blue-capped cordon-bleus and to (2) compare it to published descriptions of the song of zebra finches. Furthermore, we wanted to (3) test for sex-specific differences in solitary song and (4) to investigate whether occurrence, characteristics and performance of solitary song correlates with the level of testosterone. PLoS ONE | www.plosone.org Subjects and housing The study was conducted on eighteen adult blue-capped cordon-bleus (nine of each sex) that formed nine pairs which had at least produced fertile eggs together. Fourteen subjects (8 females, 6 males) were purchased from six different commercial breeders or pet shops and four subjects (1 female, 3 males) were reared by two different of our breeding pairs. Throughout the study, subjects were kept under non-breeding conditions, i.e. they did not have access to nesting material and were fed with nonbreeding diet (ad libitum). Non-breeding diet consists of drinking water, cuttlebone, bird’s grit and a tropical seed mixture. This was supplemented once a week with cucumber and a special food mixture which contained frosted and dried insects, egg food, dried herbs, grated carrots and apples, minerals (Supramin), micronutrients (Nektron) and vitamins (Quikon Forte). For the study, birds were housed as pairs in sound-isolation chambers measuring 70 cm650 cm and 50 cm high from inside and containing a metal wire cage equipped with three wooden perches (home cages). Birds were kept on a 14:10 light:dark schedule (lights on 07:00–21:00 Central European time) at ca. 22uC and ca. 50% humidity. Experimental procedure and sampling of blood plasma To induce solitary song (solitary-song-induced condition), the male and the female of a given pair were moved to two different sound-isolation chambers containing a cage equipped identically as the home cage (standard equipped). In the baseline condition, both members of a pair were moved together to a sound-isolation chamber containing a standard equipped cage that was not their home cage, to control for the potential confounding effect of being caught and moved into another environment. Relocation of subjects took place between 10:00 and 11:00 hours and lasted for one hour. Sixty minutes later (6062.7 min), blood samples were taken of both subjects simultaneously by wing vein puncture with a 19-gauge hypodermic needle and by collecting the blood in heparinised micro-hematocrit tubes. Afterwards, subjects were transferred into their home cages. Subsequently, blood plasma was obtained by centrifugation of whole blood and stored at 280uC upon analysis. Experiments were repeated two weeks later if (1) not enough blood plasma could be collected for the hormone assay in the first trial (11 cases), (2) no solitary song was induced in the solitary-song-induced condition (2 cases) or (3) subjects produced song in the baseline condition (1 case). Five pairs received the solitary-song-induced treatment first and four pairs received the baseline treatment first. Where experiments had to be repeated the two types of treatments were alternated. If samples of blood plasma of a given subject obtained during the first trial were too small for hormone analysis, samples from the same subject obtained during repeated trials conducted under the same condition (either baseline or solitary-song-induced) were pooled for analysis. Hormone levels revealed for such pooled samples did not differ from those of single samples (baseline condition: Mann– Whitney U test, U = 14, n1 = 13, n2 = 4, p = 0.20; solitary-induced condition: independent t-test, t = 0.54; df = 16; p = 0.60). Procedures were in accordance with national laws and approved by the Government of Upper Bavaria according to the Tierschutzgesetz (approval number 55.2-1-54-2531.3-59-07). Hormone assays Testosterone concentration was determined by direct radioimmunoassay (RIA), following [42]. Mean6SD extraction efficiency 2 October 2011 | Volume 6 | Issue 10 | e26485 Female Birdsong and Its Endocrine Correlates Figure 1. Solitary song of female and male blue-capped cordon-bleus. Each subject sang an individually characteristic song consisting of a unique sequence of syllables with some rendition to rendition variation. Sexual dimorphism in the song was subtle (cf. Table 2). Shown are images of spectral derivatives of solitary song (frequency as a function of time). The left panel (a–c) gives three examples of song renditions for each of three female subjects; the right panel (d–f) gives three examples of song renditions for each of three male subjects. Black bars on the bottom of each top image indicate syllables delineated using a 23 db threshold for amplitude and a 22.1 threshold for entropy in the features batch window of SAP. For the top images of (a) and (d), syllable labels are indicated as follows, i: introductory syllable, A, B, C etc. subsequently occurring syllable types. Note the immediately repeated syllables C, O and Q in (d). In the third rendition from the top of (f) a motif consisting of three syllables that are repeated within the same song is highlighted by a black box. See supplementary material for sound files for (a) and (d). doi:10.1371/journal.pone.0026485.g001 1000) connected to a computer. Sounds were recorded at a sampling rate of 44 kHz and 16 bit resolution using Sound Analysis Pro version 2.062 (SAP; [43]; freely available at: http:// ofer.sci.ccny.cuny.edu/html/sound_analysis.html). for plasma testosterone was 86.360.1%. All samples were analysed in one assay. Standard curves and sample concentrations were calculated with Immunofit 3.0 (Beckman Inc. Fullerton, CA), using a four parameter logistic curve fit. The lower detection limit of the standard curve was determined as the first value outside the 95% confidence intervals for the zero standard (Bmax) and was 0.41 pg/tube. The intra-assay coefficient of variation was 6.8%. Because the testosterone antibody used shows significant crossreactions with 5a-dihydrotestosterone (44%) our measurement may include a fraction of 5a-DHT. From the baseline measures of the males, one outlier (.2 SD of the mean) was excluded from the analyses (sample sizes are reduced in the relevant subset of data). Sound analysis We considered the first fifty songs (mean 6 SD mean: 50.8613) a subject produced after separation from the mate (when no song was produced in the first trial we selected songs of subsequent trials). We ignored song renditions that were preliminarily aborted by the subject and thus not performed in its full length. In a first step, we extracted several acoustic parameters using SAP. To this end, syllables were automatically delineated using a constant threshold for amplitude (23 dB) and entropy (22.1) in the features batch window (Fig. 1). These thresholds turned out to be most appropriate for this species after comparing delineation with different settings. In a next step, we printed images of spectral derivatives (representing the change of power rather than power itself, [44]) Sound recordings In each sound-isolation chamber a microphone (Earthworks TC20) was mounted on the ceiling and with a ca. 35–45u angle from the ceiling pointing to the centre of the cage. The microphone was connected to a PR8E amplifier (SM Pro Audio), which feed into an Edirol USB audio capture device (Edirol UA PLoS ONE | www.plosone.org 3 October 2011 | Volume 6 | Issue 10 | e26485 Female Birdsong and Its Endocrine Correlates frequency modulation, entropy, pitch goodness, mean frequency, [44]) and further overall and derived song features (see Table 1). For a given syllable type of a subject we averaged spectro-temporal features of all renditions of that syllable type (excluding introductory syllables). We compared differences in those features between sexes by entering values for all syllable types that a female or male subject produced into the analysis. As such values might be confounded by sex-specific differences in the repertoire composition (e.g. males could have more harmonic structures in their repertoires and therefore reach higher values of pitch goodness), we compared the repertoire compositions between females and males. As examples, we chose two characteristic classes of syllables, buzz syllables (rapid and repeated frequency modulation, with few obvious harmonics) and flat syllables (syllables containing harmonic structures also termed ‘harmonic stacks’, cf. [47,48]) and compared their proportion in the repertoires between sexes. To this end, we visually classified all types of syllables as either buzz syllables, harmonic stacks or remaining syllables following definitions of Leadbeater et al. [47] and Sturdy et al. [48]. using SAP. Based on these images and the automatic delineation of syllables we visually compared song renditions of a given subject and labelled the same syllables with the same letter according to their overall spectro-temporal appearance. This allowed us to determine the repertoire size in terms of the number of song types as well as the number of syllable types for each subject. Bluecapped cordon-bleus usually initiate a song with a variable number of introductory syllables, which were labelled as introductory syllables and not further categorized. We excluded introductory syllables from all further analysis. Cumulative curves of new syllable types plotted against the number of syllables analysed were visually checked (e.g. [45,46]). They all reached saturation, indicating that the number of songs sampled was sufficient to cover the syllable repertoire in this species. The features batch procedure in SAP provided us with parameter tables for all syllables and we manually added a column with the syllable label resulting from visual inspection. From these parameter tables and the visual inspection of images we extracted six spectro-temporal features (duration, mean pitch, Table 1. List and description of song features. Song features Description Overall song features size of song type repertoire number of different song types in the repertoire of a subject size of syllable type repertoire number of different syllable types in the repertoire of a subject % repeated syllables number of syllables that occur two or more times in a row divided by the total number of syllables song duration (ms) song duration measured from the onset of the first syllable after the introductory syllables to the offset of the last syllable as delineated in SAP number of syllables per song number of syllables per song not taken into account introductory syllables tempo (s21) number of syllables per seconds within a song increment of pitch (Hz/ms) difference in pitch from first to last syllable within a song divided by song duration correlation pitch/sequential order correlation coefficient between mean pitch of a syllable and its sequential order in the song, describes the change of pitch Spectro-temporal features (SAP) syllable duration (ms) time elapsing from the onset to the offset of a syllable mean pitch (Hz) for harmonic sounds: fundamental frequency; otherwise: mean frequency mean frequency modulation estimate of absolute slope of frequency traces mean entropy width and uniformity of power spectrum, measure of noisiness mean pitch goodness periodicity or harmonicity of the sound mean mean frequency (Hz) estimate of the centre of derivative power Spectro-temporal stereotypy min cv syllable duration minimum coefficient of variation of duration min cv mean pitch minimum coefficient of variation of pitch min cv mean frequency modulation minimum coefficient of variation of frequency modulation min cv mean entropy minimum coefficient of variation of entropy min cv mean pitch goodness minimum coefficient of variation of pitch goodness min cv mean mean frequency minimum coefficient of variation of mean frequency Sequential stereotypy internal linearity score sequential order of syllables in the song (see text) consistency score probability of consistent syllable transitions (see text) Performance related song features number of songs total number of songs in 1 h of separation (first trial) latency to sing (s) latency to sing after separation from mate (first trial) doi:10.1371/journal.pone.0026485.t001 PLoS ONE | www.plosone.org 4 October 2011 | Volume 6 | Issue 10 | e26485 Female Birdsong and Its Endocrine Correlates Figure 2. Different song types of one male blue-capped cordon-bleu. A few blue-capped cordon-bleus sang more than one song type each consisting of a unique sequence of syllables. Shown are images of spectral derivatives of solitary song (frequency as a function of time). (a) and (b) give three examples of song renditions for each of two song types sung by one and the same male. doi:10.1371/journal.pone.0026485.g002 When visually inspecting the images it seemed that subjects increase the pitch of their syllables throughout the song. To verify this observation we compared the pitch of the first and the last syllable of a given song. We were also interested whether there was a sex-specific difference in this increase and to test this we calculated the increment of pitch by dividing the difference in pitch from first to last syllable by the given song duration and we calculated the correlation coefficients between pitch and sequential order of a given syllable. As performance related song features we assessed the latency to sing in seconds from the start of separation from the mate by subtracting the time of the onset of the first song from the start of the experiment. We here only considered the first trial except for two females that did not sing solitary song during the first trial for which we took the latency of the second trial. In addition, we counted the number of songs produced during the one hour of separation (Table 1). to rendition. Consistency was calculated as the number of the most frequent transition for each syllable by the total number of transitions for this syllable. Complete consistency is represented by a score of 1, while songs with smaller scores are less consistent. Statistical analysis Statistical tests were conducted using SPSS 15.0 and R version 2.13.0 (http://www.r-project.org/). Where several measurements per subject were available we used linear mixed-effects models implemented in R2.13.0 with the add-on package nlme and entered identity of subjects as random factor. Where only one value per subject was available for a given song feature (e.g. size of repertoire) we used independent samples t-test for equality of means when testing for differences between sexes if data were normally distributed or otherwise Mann-Whitney U tests. We used Shapiro-Wilk-tests to check for normality of data. We used a paired-samples t-test when comparing the pitch of the first to last syllables (those data reached criteria for parametric testing). When testing for correlations between acoustic and hormonal data we used Pearson’s correlations tests when data where distributed normally and Spearman’s rank correlations otherwise. All tests were two-tailed and we calculated exact p values, when samples were too small for asymptotic variants of nonparametric tests [51– 53]. To control for multiple testing when comparing several song features between females and males we applied the BenjaminiHochberg false discovery rate procedure within the different categories of song features and provide adjusted P values accordingly [54]. Measuring song stereotypy We measured the song stereotypy on two different levels: (1) spectro-temporal stereotypy of syllables and (2) stereotypy of syllable sequences. To estimate spectro-temporal stereotypy we calculated the coefficient of variation (cv = SD/mean) of the spectro-temporal features provided by SAP. For reasons of comparability, we only considered the four most frequent syllables of each subject because the smallest syllable repertoire observed contained four syllables. For those syllables we selected the first 20 renditions produced after separation from the mate. For between-subjects comparisons, we choose the cv for the syllable with the lowest cv for each subject, i.e. the syllable with the lowest variability from rendition to rendition (minimum cv per subject). To estimate sequential stereotypy we calculated internal linearity and consistency following the approach described by Scharff and Nottebohm [49] and modified e.g. by Bottjer and Altenau [50]. These calculations were based on the sequences of syllable labels resulted from visual inspection of images (see above). Briefly, internal linearity was calculated as the number of different syllables produced divided by the number of different syllable-to-syllable transitions (excluding introductory syllables and transitions at the ends of songs). A song with a fixed sequence of syllables (e.g. always A-B-C-D) is perfectly linear and receives a score of 1, whereas a score close to zero indicates high variability in the sequential order from rendition PLoS ONE | www.plosone.org Results Description of blue-capped cordon-bleu song Blue-capped cordon-bleu song is initiated by introductory syllables, contains harmonic stacks and a given bird typically sings only one song type which is characterised by a unique sequence of syllables (Fig. 1, Sound file S1 and S2). However, there can be substantial intra-individual variation from rendition to rendition of a song. For instance, a part of the typical sequence can be missing in some renditions (cf. third rendition from top in Fig. 1c and first and second rendition from top in Fig. 1d) or single syllables can be repeated a different number of times (cf. Fig. 1e). Some of the subjects produced different song types (up to 4 in the current data 5 October 2011 | Volume 6 | Issue 10 | e26485 Female Birdsong and Its Endocrine Correlates Table 2. Song features in female and male blue-capped cordon-bleus. Females (n = 9) Males (n = 9) mean±SD mean±SD T 160 1.5661.01 27* df P P adjusted 0.21 0.41 Overall song features size of song type repertoirea a size of syllable type repertoire 11.8965.3 24.6767.16 24.30 16 0.001 0.004 % repeated syllablesb 15.2619.96 17.7612.32 20.39 16 0.70 0.83 song duration (ms)b 2060.266559.67 3026.761078.69 22.44 16 0.03 0.08 number of syllables per songb 10.7263.64 16.1966.39 22.40 16 0.03 0.08 tempo (s21)b 5.1461.46 5.3261.24 20.36 16 0.73 0.83 increment of pitch (Hz/ms)b 0.9760.59 0.9460.51 0.08 16 0.94 0.94 correlation pitch/sequential orderb 0.4060.27 0.4660.21 20.53 16 0.60 0.83 syllable duration (ms)b 161.75662.29 149.22626.77 0.25 16 0.81 0.81 mean pitch (Hz)b 3088.566589.08 2564.786640.34 2.00 16 0.06 0.24 mean frequency modulationb 27.1265.48 29.1566.27 20.58 16 0.57 0.70 mean entropyb 24.4260.45 23.8960.69 21.87 16 0.08 0.24 mean pitch goodnessb 153.60634.57 173.08644.41 21.12 16 0.28 0.56 mean mean frequency (Hz)b 4343.136590.67 4136.776310.50 0.57 16 0.58 0.70 1.18 16 0.25 0.46 Spectro-temporal features Measures of stereotypy Spectro-temporal stereotypy (min coefficient of variation of the relevant feature) syllable durationa a 0.0860.03 0.0660.03 0.0760.03 0.0960.04 30* 0.39 0.46 mean frequency modulationa 0.0960.02 0.0860.02 1.36 16 0.19 0.46 mean entropya 0.1460.08 0.1360.06 0.38 16 0.71 0.71 mean pitch goodnessa 0.1260.04 0.0760.02 5* mean mean frequencya 0.0360.02 0.0460.02 20.95 internal linearity scorea 0.6160.26 0.6460.18 consistency scorea 0.8660.14 0.960.07 mean pitch 0.001 0.005 16 0.36 0.46 20.24 16 0.81 0.81 20.76 16 0.46 0.81 Sequential stereotypy Performance related song features number of songsa 71666.43 151.116112.7 21.84 16 0.08 0.08 latency to sing (s)a 1005.676706.15 351.226284.69 2.58 16 0.02 0.04 a features with single values per subject analysed by independent t-test or *Mann-Whitney U in cases where data were not normally distributed. b features with multiple values per subject analysed by linear mixed-effects models. Significant p values are given in bold. doi:10.1371/journal.pone.0026485.t002 Comparison of song features between female and male blue-capped cordon-bleus set) each characterised by a unique sequence of syllables (Fig. 2, Table 2). Many subjects immediately repeated some of their syllables within a given song (5/9 females and 8/9 males, cf. Fig. 1). In addition, some subjects repeated stereotyped sequences of two or more syllables (i.e. motifs) within a given song (3/9 females and 2/9 males, cf. third rendition from top in Fig. 1f). In bluecapped cordon-bleus, pitch of syllables increased from the first to the last syllable and this difference was significant both in females (paired-samples t-test: t = 25.64, df = 8, p = 0.0005, pitch of first syllable = 22396624 Hz, pitch of last syllable = 405361095 Hz) and in males (paired-samples t-test: t = 27.79, df = 8, p = 0.0001, pitch of first syllable = 13126379 Hz, pitch of last syllable = 36096910 Hz). Note that the first syllable was, however, not necessarily the lowest-pitch syllable and the last syllable not necessarily the highest-pitched syllable in the song (cf. Table 1). PLoS ONE | www.plosone.org Males sang twice as many different syllable types than females (25 versus 12, see Table 2, Fig. 3). Also, their song tended to be longer than that of females and this seemed to be due to the larger number of syllables that males incorporated into a given song even though the difference in the number of syllables per song between females and males was not significant after correction for multiple testing (Table 2, Fig. 3). Females and males did not differ in the size of song type repertoires, the proportion of repeated syllables, the tempo and the change of pitch, both measured as the difference from last to first syllable and as correlation between pitch and sequential order of a syllable. There were no clear differences in the spectro-temporal features of females and males, i.e. none of these traits revealed a significant difference after correction for multiple testing (Table 2). However, males reached a higher level of song stereotypy with regard to pitch goodness: for 6 October 2011 | Volume 6 | Issue 10 | e26485 Female Birdsong and Its Endocrine Correlates Figure 3. Song features that differed between female and male blue-capped cordon-bleus. (a) size of syllable repertoire, (b) minimum coefficient of variation for mean pitch goodness, (c) number of syllables per song, (d) duration of song (s). For (a) and (b) single values per subject, for (c) and (d) individual means 6 SD are displayed. Note that for each feature subjects are displayed in ascending order within their sex group. Filled symbols represent females, open symbols represent males. For statistics see Table 2. doi:10.1371/journal.pone.0026485.g003 this parameter males reached a significantly lower rendition-torendition variation than females (Fig. 3). All other measures of spectro-temporal stereotypy failed to detect sex-specific differences in the song of blue-capped cordon-bleus, and females and males did also not differ with regard to the sequential stereotypy (Table 2). The sexual dimorphism in song stereotypy with regard to pitch goodness might be related to a higher abundance of harmonic stacks in the song of males. To rule out this possibility, we compared the proportions of harmonic stacks and another characteristic class of syllables, the buzz syllables (rapid and repeated frequency modulation, with few obvious harmonics) in the repertoires of females and males. Harmonic stacks made up 24.5623.5% of syllables in the repertoires of females and 29.2610.4% in the repertoires of males and this proportion was not different (independent samples t-test: t = 20.55, df = 16, p = 0.59). Buzz syllables made up 13.761.3% of syllables in the repertoires of females and 11.260.3% in the repertoires of males and this proportion was also not different (independent samples ttest: t = 0.56, df = 9, p = 0.59). In addition, we measured two performance related song traits and compared them between the sexes: males tended to sing more songs during one hour of separation from the mate and they started singing earlier after their mate had been removed (Table 2). testosterone between males and females in both conditions (baseline: independent samples t-test: t = 21.44, df = 15, p = 0.17; solitary-song-induced: independent samples t-test: t = 21.33, df = 16, p = 0.20) we pooled data of males and females to compare between the different treatment conditions. Birds had significantly higher levels of testosterone in the solitary-induced condition than in the baseline condition (mean 6 SD mean: 375.296204.70 versus 239.326103.20 pg/ml, paired-samples ttest: t = 23.05, df = 16, p = 0.008, Fig. 4) and when testing the sexes separately a trend remained for both of them (paired-samples t-test: females t = 22.02, df = 8, p = 0.08; males: t = 22.22, df = 7, p = 0.06, Fig. 4). Reanalysing these data including the one outlier (one male had a level of testosterone of 1767.84 pg/ml in the baseline condition) did not change the results except that males’ levels of testosterone in the reanalysis clearly did not differ between baseline and solitary-induced condition when tested separately. Furthermore, we compared levels of testosterone in the solitarysong-induced condition with performance related song features. Testosterone did not correlate with the number of songs produced in the one hour of separation (Pearson’s correlation test: r = 0.27, N = 18, p = 0.28). Visual inspection of the graphs plotting latency to sing against the concentration of testosterone suggested a trend in females, but the correlation just failed to be significant (Pearson’s correlation test: r = 20.65, N = 9, p = 0.06; Fig. 5). In males, no such trend could be observed (Pearson’s correlation test: r = 20.15, N = 9, p = 0.69; Fig. 5). Thus, those blue-capped cordon-bleu females with higher levels of testosterone tended to sing earlier after their mate was removed, besides that all males started to sing early. However, when analysing data of females and Endocrine correlates of solitary song To investigate whether the occurrence of solitary song was correlated with an increase of testosterone, we first compared the level of testosterone during baseline condition and during solitarysong-induced condition. As there were no differences in the level of PLoS ONE | www.plosone.org 7 October 2011 | Volume 6 | Issue 10 | e26485 Female Birdsong and Its Endocrine Correlates Figure 5. All males and females with high testosterone levels sang rapidly after separation from their mate. Latency to sing solitary song (s) as a function of testosterone concentration (pg/ml) in the blood plasma. Filled symbols and the solid line represent females; open symbols and the broken line represent males. doi:10.1371/journal.pone.0026485.g005 Figure 4. Testosterone was higher in the solitary-song-induced condition compared to the baseline condition. Testosterone concentration (pg/ml) in the blood plasma collected after one hour of baseline treatment (i.e. being transferred into another sound-isolation chamber together with the mate) and one hour of solitary-songinduced treatment (i.e. being transferred into another sound-isolation chamber without the mate). Filled symbols represent females, open symbols represent males. doi:10.1371/journal.pone.0026485.g004 linearity score of 0.95 and a consistency score of 0.96 for zebra finch undirected song [50]. Blue-capped cordon-bleus belong to the same family (Estrildidae) as the zebra finch. It is therefore not surprising that they share some song features with zebra finches: for instance, in both species, song is mainly produced in two different social contexts, and a given bird normally sings only one individually distinct song type. Another typical feature of both species is that the song contains multi-component harmonic structures. Like zebra finches, bluecapped cordon-bleus show the phenomenon of immediate syllable repetition within a given song, however, such repetition occurred much more often in our subjects than in zebra finches. Most of the spectro-temporal features vary between the two species: blue-capped cordon-bleus sang at higher pitch and their songs were shorter than those of zebra finches. Their song reached higher levels of pitch goodness than that of zebra finches, i.e. contained a higher proportion of energy in multi-component harmonic structures. Frequency modulation was similar in both species but entropy was much higher in zebra finches than in bluecapped cordon-bleus, indicating that the song of blue-capped cordon-bleus is less noisy than the song of zebra finches. In the majority of blue-capped cordon-bleus (72%) the song was not organised in motifs, that is, birds produced the sequence of their syllables only once within a song. In this respect, song organisation varies from that of zebra finches, which repeat stereotyped sequences of syllables (i.e. motifs) several times within a song. Overall, the sequential order of syllables was more variable in blue-capped cordon-bleus than in zebra finches and they were also less consistent in following particular sequences. The most striking difference between the species is that in zebra finches only males sing, whereas in blue-capped cordon-bleus both females and males sing – the reason for us to establish them as a new model species. males together, the relationship between testosterone and latency to sing becomes significant (Pearson’s correlation test: r = 20.49, N = 18, p = 0.04). Thus, further studies are needed to confirm that this is an effect in females only. Finally, we compared levels of testosterone in the solitary-song-induced condition (pooled for both sexes) with overall song features, spectro-temporal features and stereotypy. None of these parameters correlated with the level of testosterone (Spearman’s rank or Pearson’s correlations test: overall song features: |r|,0.32, N = 18, p.0.19; spectro-temporal features: |r|,0.37, N = 18, p.0.12; spectro-temporal stereotypy: |r|,0.25, N = 18, p.0.30, sequential stereotypy: |r|,0.18, N = 18, p.0.47). Discussion Comparison with zebra finch song Zebra finch song consists of a series of introductory syllables followed by two to four repetitions of an individually distinct motif, which is a stereotyped sequence of several (usually four to seven) distinctive syllables. Songs are usually produced in bouts with each male singing an individually distinct song [16,55]. A characteristic feature of zebra finch syllables is that they often contain multicomponent harmonic structures. Infrequently, zebra finches immediately repeat a given syllable within a motif, such immediate repetition has only been described for captive birds and amongst them only in a small fraction (7%, [56]). The duration of undirected song in zebra finches is on average 5170 ms [57]. Mean pitch of undirected song lies between 983 and 1092 Hz. Pitch goodness, a measure for how much energy lies in harmonics, reaches levels from 35 to 38. Mean values of frequency modulation are 28 to 31 and entropy, a measure for noisiness, adopts values between 22.9 to 22.5 (all measured for undirected song, [58]). Finally, measures for sequential stereotypy reveal an internal PLoS ONE | www.plosone.org Comparison of female and male song in blue-capped cordon-bleus Syllable repertoire size was twice as large in males and thus males reached a higher level of song diversity than females. Also the song of males tended to be longer in duration. This is in line with findings on some but not all other studied species, in which 8 October 2011 | Volume 6 | Issue 10 | e26485 Female Birdsong and Its Endocrine Correlates of the mate) could lead to higher levels of testosterone on one hand and to higher song output on the other. The finding that those females that sang earlier tended to have higher testosterone levels, suggests a causal relationship between the hormone level and solitary song at least in females, although given that this correlation becomes significant only if females and males are analysed together, further studies would be needed to confirm that this is an effect in females only. A causal relationship between testosterone and the onset of directed song in male zebra finches has been demonstrated experimentally: males that had been implanted with testosterone started to produce directed song earlier after the introduction of a female than before the implantation [69]. Whilst the authors in [69] measured this effect only seven days after the implantation, in principle, steroid hormones can fluctuate in a rapid time course depending on the social context [70]. Furthermore, hormone treatment can influence vocal behaviour within a few minutes, as has been shown for structural vocal parameters in midshipman fish (Porichthys notatus, [71]). Thus, it is conceivable that the described increase in testosterone facilitates singing of solitary song in bluecapped cordon-bleus, i.e. small increases in testosterone levels would have large behavioural consequences. Alternatively, there could be a causal link between hormone and behaviour in the opposite direction. Thus, the increase of singing could lead to an increase in testosterone and therefore females that start to sing earlier would have higher hormone levels. Such a relationship has been demonstrated for female ring doves (Streptopelia risoria) in which nest cooing vocalisations stimulate the release of hormones that lead to follicular growth and ovulation (reviewed in [72]). Experiments manipulating the hormone levels will be needed the fully understand the relationship between testosterone and singing in blue-capped cordon-bleus. Testosterone levels of blue-capped cordon-bleus were low in comparison to male zebra finches [66] but within the range of other tropical passerines. Non-breeding testosterone concentrations of 33 tropical species was (mean 6 SD) 421.86428.3 pg/ml (calculated from testosterone concentrations reviewed in [73]). Thus, blue-capped cordon-bleus fit in the general pattern of tropical species having low testosterone concentrations ([1], but see [74]), a phenomenon that might be associated with tropical life history traits such as year-round territoriality and asynchronous breeding [74,75] or with an immune system function related to greater pathogen burden in the tropics [76]. On a more proximate level, low levels of testosterone might be related to an increase in target sensitivity e.g. by increasing expression of androgen receptors in relevant brain regions [77,78]. females regularly sing. In female black-bellied wrens for instance, songs were shorter and their repertoires smaller than in males [22] and in red-cheeked cordon-bleus, songs of females were shorter than that of males [20]. Likewise, zebra finch females that had received an estradiol-treatment as hatchlings to masculinise their song system produced shorter songs than males as adults [39]. In other, mostly duetting, species both sexes have been reported to reach the same level of song diversity (e.g. bay wren, Thryothorus nigricapillus, [59]; forest weaver, [60]; white-browed sparrow weavers, Plocepasser mahali, [61]). The diversity of female relative to male song forms a continuum between species with male-only song production on one end and species with equality of song diversity on the other [62]. Blue-capped cordon-bleus seem to be intermediate between these two extremes. In addition to song diversity, we found another clear sexual dimorphism in the rendition-to-rendition variation of pitch goodness: males reached a lower level of variation in the pureness of multi-component harmonic structures. Thus, their song was more stereotyped with respect to this parameter and since males did not have more harmonic stacks in their repertoires than females, this difference was not due to differences in the repertoire composition. All the other measurers of stereotypy did not differ between females and males. Thus, in comparison to other species, in which females clearly show a higher level of song variability (e.g. canaries, Serinus canaria, [63], Northern cardinal, Cardinalis cardinalis, [64]) differences in song stereotypy in blue-capped cordon-bleus seem rather subtle. Sexes did, however, vary in performance related song features: males tended to sing more songs and started to sing earlier during the one hour of separation. Besides these dimorphisms in syllable repertoire, song duration, pitch goodness and the performance related features, all of the remaining song features did not differ significantly between the sexes. In the current study we did not compare song features between members of a pair-bond. Blue-capped cordon-bleus would, however, provide a perfect model to investigate whether vocal convergence occurs between partners such as has been described for instance in budgerigars (Melopsittacus undulates, [65]). Endocrine correlates of solitary song The overall plasma testosterone concentrations of blue-capped cordon-bleus were similar in both sexes. This is in contrast to findings in zebra finches were males have higher levels of testosterone (mean 6 SD mean: 14346126 pg/ml, Table 5 in [66]) than females (mean 6 SD mean: 4006438 pg/ml, Fig. 1 in [67]) and even in Northern cardinals, another species with female and male song production, males had slightly higher levels of testosterone than females (mean 6 SE: 18326130 pg/ml versus mean 6 SE: 1440638 pg/ml, [68]). In blue-capped cordon-bleus, the fact that both females and males sing is paralleled by the lack of a sexual dimorphism in testosterone profiles. Testosterone levels in cordon-bleus did also not correlate with the subtle sexual dimorphic song motor pattern. Thus, neither any overall song feature (such as repertoire size, song duration etc.) nor any spectrotemporal feature or song stereotypy correlated with the level of testosterone. Also the number of songs produced did not correlate with testosterone, and this is in contrast to findings on male zebra finches, in which such a correlation was demonstrated [66]. Although testosterone levels do not explain sex differences in song pattern, they might be related to singing activity. Despite the relatively low levels of testosterone in blue-capped cordon-bleus, solitary song occurred in parallel to an increase in testosterone. The correlative nature of this finding does not allow us to rule out the possibility that a third factor (e.g. being stressed by the absence PLoS ONE | www.plosone.org Supporting Information Sound File S1 Solitary song of a female blue-capped cordon-bleu. An image of the spectral derivative of this song is shown in the top of Figure 1a. (WAV) Sound File S2 Solitary song of a male blue-capped cordon-bleu. An image of the spectral derivative of this song is shown in the top of Figure 1d. (WAV) Acknowledgments We are grateful to Lisa Trost for help with running the experiments, and to Wolfgang Goymann, Ingrid Schwabl and Monika Trappschuh for the hormone analysis. Many thanks to Roswitha Brighton, Rositta SiegelHirner and David Witkowski for taking care of the birds and to Dieter Schmidl and Gerhard Hofmann for advice on breeding them. We thank 9 October 2011 | Volume 6 | Issue 10 | e26485 Female Birdsong and Its Endocrine Correlates Mihai Valcu for advice on statistics and Gabriel Beckers, Sebastien Derégnaucourt, Wolfgang Goymann, Albertine Leitão, Ali Steinbrecht and two anonymous reviewers for comments on a draft version of this manuscript. Sue Anne Zollinger deserves thanks for improving the English of the manuscript. 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Behav Ecol Sociobiol (2006) 60: 234–241 DOI 10.1007/s00265-006-0161-9 ORIGINA L ARTI CLE William A. Searcy . Rindy C. Anderson . Stephen Nowicki Bird song as a signal of aggressive intent Received: 25 August 2005 / Revised: 25 November 2005 / Accepted: 9 January 2006 / Published online: 24 February 2006 # Springer-Verlag 2006 Abstract A central question in animal communication research concerns the reliability of animal signals. The question is particularly relevant to aggressive communication, where there often may be advantages to signaling an exaggerated likelihood of attack. We tested whether aggressive signals are indeed reliable signals of attack in song sparrows (Melospiza melodia). We elicited aggressive signaling using a 1-min playback on a male’s territory, recorded the behavior of the male for 5 min, and then gave him the opportunity to attack a taxidermic mount of a song sparrow associated with further playback. Twenty subjects attacked the mount and 75 did not. Distance to the speaker was a significant predictor of attack for both the initial recording period and the 1 min before attack. For the initial recording period, none of the measures of singing behavior that we made was a significant predictor of attack, including song-type matching, type-switching frequency, and song rate. For the 1-min period immediately before attack, only the number of low amplitude “soft songs” was a significant predictor of attack. Although most aggressive signals contained little information on attack likelihood, as some models suggest should be the case, the unreliability of these signals was not caused by convergence of individuals on a single signaling strategy, as those models argue should occur. Keywords Communication . Bird song . Aggressive signals . Reliability . Melospiza melodia Communicated by J. Podos W. A. Searcy (*) . R. C. Anderson Department of Biology, University of Miami, Coral Gables, FL 33124, USA e-mail: wsearcy@miami.edu Fax: +1-305-2843039 S. Nowicki Department of Biology, Duke University, Durham, NC 27708, USA Introduction The reliability of animal signals is a major issue in research on animal communication (Maynard Smith and Harper 2003; Searcy and Nowicki 2005). The issue is particularly critical with respect to aggressive signaling. Honest signaling provides a cheap and efficient mechanism for settling aggressive encounters, but a signaling system that is honest about aggressiveness is vulnerable to a form of cheating in which individuals exaggerate their aggressiveness to win contests that they would otherwise lose (Maynard Smith 1974, 1979; Dawkins and Krebs 1978). Game theoretical models have shown that signals can be honest about aggressive intentions under certain conditions, for example if the most aggressive and effective signals are also the most costly (Enquist 1985; Grafen 1990). Even in such models, the power of an aggressive display to predict a signaler’s behavior may be modest, for example because the behavior of the signaler after a signal is contingent on external factors such as the receiver’s response (Enquist 1985). Moreover, other models have shown that aggressive signaling systems can be evolutionarily stable with a mix of honest and dishonest signals, with some individuals benefiting from producing signals that exaggerate their aggressive intentions (Adams and Mesterton-Gibbons 1995; Számadó 2000). Thus, theory allows a range of outcomes with respect to reliability in aggressive signals, and empirical investigation is needed to establish the true state of affairs with respect to a specific signaling system. In this study, we describe an experimental investigation of the reliability of aggressive vocal signals in song sparrows (Melospiza melodia). Song sparrows exhibit a number of the singing behaviors that have been hypothesized to be aggressive signals. Among these is song-type matching, a behavior in which an individual replies to a rival with a song type that is identical (or highly similar) to the one a rival has just sung. Krebs et al. (1981) suggested that matching is a graded signal of aggressiveness, in the sense that “a territory holder signals the likelihood of attacking an intruder by the probability of matching the intruder’s song.” In support of this view, Krebs et al. (1981) 235 demonstrated in great tits (Parus major) that a correlation exists between percent matching to playback and indirect measures of aggression such as latency to respond and close approach to the speaker. In song sparrows, Vehrencamp (2001) showed that males that matched a playback song spent more time close to the speaker than males that did not match; again, time spent close to a playback speaker is usually assumed to indicate aggressiveness. Beecher et al. (2000), however, found no difference in aggressiveness between male song sparrows that did or did not type match, with aggressiveness measured by a combination of approach and display. To our knowledge, no one previously has tested whether matching predicts actual attacks, as we do in this study. Another signaling option available to songbirds with repertoires of song types is to vary the frequency of switching between types. The frequency of switching increases in aggressive contexts in a number of species of songbirds (Falls and D’Agincourt 1982; D’Agincourt and Falls 1983; Simpson 1985), including song sparrows (Kramer and Lemon 1983; Kramer et al. 1985; Searcy et al. 2000), suggesting that rapid switching is an aggressive signal. Type-switching frequencies did not, however, predict attack on a caged intruder in male bobolinks (Dolichonyx oryzivorus) (Capp and Searcy 1991). Song sparrows also possess another level of song variation, song-type variants, which are relatively minor variations of individual song types. Although the differences between song variants are small compared to the differences between song types (Podos et al. 1992), male song sparrows are responsive to this level of variation (Stoddard et al. 1988; Searcy et al. 1995). In aggressive contexts, song sparrows increase variant-switching frequencies even more consistently than they increase type-switching frequencies (Searcy et al. 2000). Thus, variant switching may also be an aggressive signal. The rate at which songs are sung is a third aspect of singing behavior that has been suggested to be a signal of attack likelihood (Falls 1969). Song rates depend on male condition in many songbirds (Davies and Lundberg 1984; Gottlander 1987), and we might expect aggressiveness also to covary with condition. Song rates have been shown to increase in aggressive situations in some songbird species (Falls 1969), but such an increase does not occur reliably in song sparrows (Peters et al. 1980). A final singing behavior that might convey aggressiveness is the production of low amplitude “soft song.” Low amplitude songs have been noted in several songbird species as occurring during aggressive encounters, courtship, or both (Dabelsteen et al. 1998). In song sparrows, soft song is typically observed only during aggressive encounters (Nice 1943). The association of particular singing behaviors with aggressive situations suggests that these behaviors are signals that convey aggressive intent, but taken by itself, such an association is equally compatible with the displays having a submissive meaning. Submissive displays, like aggressive ones, are given during agonistic encounters rather than during foraging, resting, and so forth, and thus their frequencies also can be expected to increase during encounters relative to their background frequencies. To support the interpretation that a signal conveys aggressive intent, it is necessary to demonstrate not merely that the signal is produced in an aggressive context, but that the signal actually predicts subsequent aggressive behavior by the signaler. Tests of this more stringent criterion must overcome several methodological challenges. Much of the early work on the reliability of aggressive signals focused on avian postural displays given during contests over food. These studies concluded that some displays do predict aggression (Stokes 1962; Dunham 1966; Andersson 1976), but Caryl (1979) criticized the evidence on the grounds that the associations between displays and aggression were generally quite weak. One explanation for the weakness of the associations found in these studies is that all were observational studies of natural interactions, which looked at whether a signal was followed by a certain signaler behavior without controlling for the response of the receiver. One way around this problem is to use a statistical analysis that controls for receiver’s response (Popp 1987); another is to replace the natural receiver with an experimental stimulus whose behavior can be controlled. This latter approach has been taken by experiments in which aggressive displays are elicited by playback of vocal signals, for example in frogs (Wagner 1992; Burmeister et al. 2002) and birds (Vehrencamp 2001). Playback studies typically introduce a new problem, however, in that outright attacks are no longer possible, so that one must examine associations between display and some weaker proxy of aggressive response, such as approach to the speaker. A final problem seen in some studies of aggressive communication is that the periods in which display and aggressive acts are measured may be partially or totally overlapping, so that some of the displays may be given after the aggression that they “predict” (e.g., Krebs et al. 1981; Husak 2004). In our work, we employed a design that obviates these problems: we used an artificial stimulus to remove the problem of contingencies based on receiver’s response; we provided a taxidermic model as part of the stimulus so that outright attacks would be possible; and we maintained a temporal separation between signals and aggression so that displays we analyzed would always precede the behavior that they predicted. Materials and Methods Playback experiments Playback trials were carried out in Crawford County, Pennsylvania, U.S.A. during May and June of 2000, 2001, and 2002, between the hours of 6:00 and 11:00 AM. Each subject was a territorial male song sparrow that had previously been recorded; no male was tested more than once. We used one of the subject’s own song types in constructing each playback tape so that the subject could match the playback if he chose to do so. Recordings were made using a Realistic omnidirectional microphone in a 236 Sony PBR-330 parabola and either a Sony TCM 5000 or a Sony TC D5M tape recorder. Choice of the one song type to be used in playback for each subject was made on quality of recording. Playback songs were digitized at 25 kpts/s using ‘SIGNAL’ software (Engineering Design, Berkeley, CA, USA) and then dubbed onto a Sony TCM 5000 tape recorder. Before a trial, we placed a Nagra DSM speaker face up on the ground well within the subject’s territory. On top of the speaker, we placed a stand holding a taxidermic mount of a song sparrow, posed in a normal, perched posture. The mount was concealed by a cloth at the start of the trial. A microphone was positioned 10–15 cm from the mount and was connected via cable to one channel of a Sony TC D5M stereo tape recorder. This recorder was operated by one of two observers, who stood 15–20 m from the speaker/mount setup; this observer also held a Sony PBR-330 parabola with a Realistic omnidirectional microphone plugged into the second channel of the stereo recorder. The second observer narrated the behavior of the subject throughout the trial, and this commentary was recorded, along with the bird’s vocal behavior. The narration included estimates of the subject’s distance from the speaker, aided by markers at 0.5, 1 m, 4, and 8 m on either side of the speaker. The narration also noted bouts of wing waving, a stereotypic wing movement used as a postural display in aggressive contexts by song sparrows and other passerines (Nice 1943). Finally, the observer classified all songs produced by the subject as loud or soft. We subsequently tested the accuracy of the same observer (WAS) in making amplitude judgments, using playback of a variety of song sparrow song types at predetermined amplitudes that ranged in 3 dB intervals from 89 to 57 dB SPL (measured at 1 m with a GenRad 1565D sound level meter). The observer was tested in the field, in the same habitat used in the trials, and at the same observation distance. Seventyeight percent of songs played at 78–80 dB and 100% of songs played at ≥80 dB were classified as loud, whereas 74% of songs played at 75–77 dB and 100% of songs played at ≤74 dB were classified as soft. A trial began with 1 min of playback of the subject’s chosen song type, presented at a rate of one song per 10 s and at an intensity of approximately 85 dB SPL measured at 1 m with a GenRad 1565-D sound level meter. The first playback song was used to mark the beginning of the trial; recording began some seconds before this. At 5 min and 45 s, the mount was revealed by removing the cloth cover remotely. At 6 min, a second playback bout began, using the same playback song, presented at the same rate but at a lower amplitude (approximately 79 dB SPL at 1 m). This second playback lasted 2 min. The subject was given until 20 min into the trial to attack the mount. An attack was considered to be any direct physical contact of the subject with the mount; sometimes, this contact consisted of a brief pounce, but more typically a subject landed on the mount and pecked vigorously at its head and body. Trials were ended at the point of attack. We continued recording until an attack occurred or 20 min was reached, whichever came first. Analysis The entire narration of each trial was transcribed onto a flow sheet divided into 10 s intervals. Subsequent analysis concentrated on two time periods: the initial recording period and the 1 min before attack. The initial recording period began with the first playback song and lasted for 5 min. For those subjects that attacked, the 1 min before attack was the 1 min preceding the 10 s interval in which the attack occurred. For those subjects that did not attack, a corresponding 1-min period was chosen by matching each of the 20 attackers to three to four of the 75 non-attackers drawn randomly without replacement, and analyzing the same 1-min period in the non-attacker as in the matched attacker. We compared the resulting 75 1-min periods for non-attackers to the 20 1-min periods for attackers in an unpaired analysis, thus using each subject once in the analysis. All songs recorded during the initial recording period and the 1 min before attack were digitized and viewed as spectrograms using Syrinx sound analysis software (J. M. Burt; http://syrinxpc.com). From the spectrograms, we determined for each song whether it (a) matched the playback song type, (b) represented a change in song type relative to the preceding song produced by the subject, and (c) represented a change in song-type variant relative to the preceding song. We also made spectrograms of enough songs produced by the subject after the onset of the second playback to determine whether the subject matched that playback with the first song he produced or the first song type to which he switched. The random expectation of matching was assumed to be 12.5%, the probability of meeting the matching criteria found in previous control trials with this population (Anderson et al. 2005). From the spectrograms and narration, we abstracted six measures of display for both the initial recording period and the 1 min before attack. These measures were: (1) matches—the number of songs that matched the playback type; (2) soft songs—the number of soft songs produced by the subject; (3) total songs—the total number of songs, including matches and soft songs; (4) type-switching frequency—the number of type switches divided by the number of opportunities to switch types (Searcy et al. 2000); (5) variant-switching frequency—the number of variant switches divided by the number of opportunities to switch variants (Searcy et al. 2000); and (6) wing waves—the number of bouts of wing waving. Sample sizes are slightly smaller for results on the two switching variables, which could not be calculated in cases in which the denominator (opportunities to switch) was 0. Mean distance to the speaker during a period was estimated by first calculating the mean of the estimates made within each 10-s block and then averaging across all 10-s blocks. Several of the response variables were not normally distributed and could not be normalized using standard transformations. We therefore first analyzed each variable by comparing attackers to non-attackers using a nonparametric procedure, the Mann Whitney U test. We also analyzed the response variables, separately and together, with discriminant function analysis, because this procedure 237 allows the possibility of discriminating between groups using combinations of variables. Discriminant function analysis assumes a multivariate normal distribution but is considered to be robust against departures from this assumption (Klecka 1975). We do not consider a result to be significant unless significance is supported by both the parametric and nonparametric tests. Because we use six different display measures to test the general hypothesis that display predicts attack, we use a Dunn–Sidák-corrected alpha level of 0.0085 for those tests. Results Mean distance to the speaker during the initial recording period was lower for attackers (4.3±0.5) than non-attackers (8.4±0.7), and this difference was significant by a Mann Whitney U test (P=0.005). Mean distance to the speaker also was a significant predictor of attack in a discriminant function analysis (Wilks’ lambda=0.907, F=9.58, N=95, P=0.0026). In the jack-knifed classification matrix, distance to speaker correctly predicted 16 of 20 attacks (80%) and 44 of 75 failures to attack (59%). In a forward stepwise discriminant function analysis, no display variable entered after distance, that is, none had additional predictive power once distance was taken into account. Initial recording period One minute before attack Of the 95 males that we tested, 20 attacked the mount within the allotted time and 75 did not. For the 5-min initial recording period, none of the six display measures differed significantly between attackers and non-attackers according to Mann Whitney U tests (Fig. 1). When entered separately into discriminant function analyses with attack as the categorical variable, none of the display measures was a significant predictor of attack. The model with number of soft songs had a low P value (Wilks’ lambda=0.951, F=4.75, N=95, P=0.032) but not lower than the Dunn–Sidák-corrected alpha value. For the 1 min before attack, the number of soft songs was significantly higher for those males that attacked than for those that failed to attack (Fig. 2a), and this difference remained significant with the Dunn–Sidák correction. None of the other five display measures differed significantly between attackers and non-attackers (Fig. 2b–f). In single variable discriminant function analyses, the number of soft songs was the only display variable that significantly predicted attack (Wilks’ lambda=0.912, F=8.95, N=95, P=0.0035). In the jack-knifed classification matrix, the number of soft songs correctly predicted 12 of 20 attacks Fig. 1 Displays given during the initial 5-min recording period by those male song sparrows that subsequently did or did not attack the mount. a Low amplitude “soft songs” [Mann Whitney U=540.5, N1(attackers)=20, N2(non-attackers)=75]. b Type-switching frequen- cy (U=814.5, N1=20, N2=74). c Variant-switching frequency (U=840, N1=20, N2=74). d Total songs (U=543, N1=20, N2=75). e Matching songs (U=687, N1=20, N2=75). f Bouts of wing waving (U=610.5, N1=20, N2=75). Histograms show means±SE 238 Fig. 2 Displays given during 1-min period before attack by those male song sparrows that subsequently did or did not attack the mount. a Low amplitude “soft songs” [Mann Whitney U=370.5, N1(attackers)=20, N2(non-attackers)=75]. b Type-switching frequen- cy (U=619, N1=20, N2=60). c Variant-switching frequency (U=416, N1=20, N2=57). d Total songs (U=574, N1=20, N2=75). e Matching songs (U=685, N1=20, N2=75). f Bouts of wing waving (U=561, N1=20, N2=75). Histograms show means±SE (60%) and 58 of 75 failures to attack (77%). None of the other display measures was a significant predictor of attack when entered alone, nor did any of these measures provide significant additional predictive power when entered in a forward discriminant function analysis together with the number of soft songs. Mean distance to the speaker during the 1 min before attack was lower for attackers (1.8±0.8) than for nonattackers (8.0±1.1), and this difference was significant by a Mann Whitney U test (P=0.00021). Distance during the 1min periods was also a significant predictor of attack in a discriminant function analysis (Wilks’ lambda=0.916, F=8.58, N=95, P=0.0043). The P value, however, was not as low as for soft songs, and when both distance and soft songs were entered in a forward stepwise discriminant function analysis, only the soft song variable was retained in the model. matches the playback. Using these criteria, 41 of 95 subjects (43%) matched the first playback, significantly more than expected by chance (X2=81.4, df=1, P<0.0001). However, the percentage of matchers attacking (19.5%) was not higher than the percentage of nonmatchers attacking (22.2%). Only 11.6% (11 of 95) of the subjects matched the second playback, quite close to the random expectation of 12.5%. The percentage of matchers attacking (18.2%) was again not higher than the percentage of nonmatchers attacking (21.4%). Matching Song-type matching is often treated as a categorical variable (males match or do not match), rather than as a continuous variable as above. The criterion usually applied in a categorical analysis is that a subject is considered to have matched if the first song type he produces after the playback starts, or the first song type he switches to Discussion The vocal and visual displays produced by song sparrows during an aggressive encounter for the most part contained little or no information on the signaler’s likelihood of attack, even though each of the displays has been labeled at one time or another as an aggressive signal. Proximity to the stimulus did consistently predict attack across both recording periods; this result has methodological implications, as it supports the widespread practice of using distance to a loudspeaker or other stimulus as a proxy for aggressiveness. Proximity cannot, however, be considered a signal if signals are defined as “characteristics fashioned or maintained by natural selection because they convey information to other organisms” (Otte 1974). In other 239 words, position of one individual relative to a second may convey information to the second, but position is not a characteristic that has evolved to convey that information. The conclusion that most signals, given in an aggressive context, do not contain information about attack seems particularly surprising with respect to type matching, given that song-type matching has been shown to be associated with correlates of aggression such as time of season and approach to a perceived intruder in both song sparrows (Beecher et al. 2000; Vehrencamp 2001) and other species (Krebs et al. 1981). The explanation may be that type matching is a signal of attention rather than aggression, as originally suggested by Bremond (1968), and is predictive of milder forms of escalation than attack (Burt et al. 2001). The one display that we found to contain reliable information about attack likelihood is low amplitude soft song. Soft song was not only a better predictor of attack than any of the other displays we observed, but it was also a better predictor than proximity during periods immediately before attack. Soft song was in general associated with close proximity, but we also observed soft song produced as far as 30 m from the mount; conversely some males gave broadcast song from within 0.25 m of the mount, or approached the mount closely while not singing at all. Thus, soft song production does provide information independent of proximity. Soft song may seem an odd signal to use to convey the highest level of aggressive intent, in that the signal’s form (i.e., its low amplitude) appears to be antithetical to its function (intimidating rivals). One explanation for the use of soft song in aggressive encounters is that the low intensity of the signal may benefit the signaler by minimizing eavesdropping (McGregor and Dabelsteen 1996). McGregor (1993) defines eavesdropping as occurring when “individuals not directly involved in a signaling interaction... none the less gather information from it.” Recent studies demonstrate that eavesdropping in this sense occurs in territorial birds and other animals (Oliveira et al. 1998; Otter et al. 1999; Peake et al. 2001). Territorial males that use low amplitude songs when confronting intruders might benefit through concealing such interactions from eavesdropping females, who might lower their assessment of males that have difficulties with intruders, and from eavesdropping males, who might be tempted to trespass on territories whose owners appear to be preoccupied already with an intruder. We have recently shown, however, that neighboring males do not trespass less when we simulate an interaction between a soft owner and a loud intruder than when we simulate a loud owner/loud intruder interaction (Searcy and Nowicki unpublished). Despite our finding that soft song predicts attack, the overall impression from our results is that display behavior in song sparrows provides surprisingly little information on attack likelihood. This conclusion is consistent with theoretical predictions made by Maynard Smith (1974, 1979) and Caryl (1979), but our results do not necessarily support their reasoning. These authors suggested that signals would contain little information on attack likelihood because most or all signalers would converge on the most aggressive signaling pattern, which would be the most effective behavior in terms of winning encounters. Our observations provide little evidence for such convergence. What we observed instead was enormous variability in the signals given among the males that attacked as well as among those that did not. Variability in display by those that do not attack can be explained by the hypothesis that some individuals follow a dishonest signaling strategy of exaggerating their aggressiveness; variability in display by those that do attack requires a different explanation. Another explanation for why signals produced in an aggressive context are poor predictors of attack invokes the role of contingency. By contingency, we mean the dependence of the signaler’s behavior on events that occur in the time interval between the signal and the subsequent behavior. Effects of contingency are built into many signaling models; for example, in the first model of Enquist (1985), a signal that is completely honest about the intention to attack is followed by attack in only half the instances it is given, because in the other half, the receiver submits immediately, making an attack unnecessary. In our experiments, we minimized the influence of this type of contingency by employing an inanimate opponent whose behavior did not vary between trials. Other types of contingencies are still possible, however; for example, a signaler might give an aggressive signal and then, before it can attack, be distracted by the behavior of its mate, a territorial neighbor, a predator, and so forth. The fact that in our results attack was better predicted by signals given in the 1 min just before attack than by signals given earlier (in the initial recording period) is consistent with the contingency hypothesis, as the shorter the interval between signal and subsequent behavior, the less opportunity there is for contingency to intervene. As with the dishonest signaling hypothesis, however, contingency seems a better explanation for why some individuals give aggressive signals and then fail to attack, than of why some individuals attack without giving aggressive signals beforehand. In previous work, we have found that territorial male song sparrows show considerable variation in aggressive and singing rates in response to intrusions, and that individuals are consistent from trial to trial in these behaviors (Nowicki et al. 2002; Hyman et al. 2004). Individuals are also consistent between trials in the proportion of soft songs that they produce (Searcy and Nowicki unpublished). Individual behavior is consistent across trials despite the contingencies that arise in successive trials, implying that contingencies cannot be blamed entirely for the weakness of the associations between signaling and subsequent aggression. Individual strategies seem to include all possible combinations of weak signaling/weak aggression, strong signaling/weak aggression, weak signaling/strong aggression, and strong signaling/strong aggression. Despite the variability in individual strategies, almost all males are able to maintain their territories, which suggests that the fitness payoffs of the different strategies are similar. We suggest that multiple strategies of equal overall fitness exist for both signaling and aggression, and that the equivalence of these strategies is one major reason that the 240 relationship between signals and subsequent aggression is inherently weak. Acknowledgements We thank the Pennsylvania Game Commission and Pymatuning State Park for the access to study sites, and the Pymatuning Laboratory of Ecology, University of Pittsburgh, for the use of laboratory facilities. Financial support was provided by grants from the National Science Foundation to W.A.S. (IBN-0315566) and to S.N. (IBN-0315377). This research complies with the laws and regulations of the U.S.A. and Pennsylvania. 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Reference # “Literature Review Detailed Summary” 1. Citation of the paper that is being described: 2. What is the main hypothesis or idea being examined in this study? What is the hypothesis of the study? Do not copy and paste, re-write it 3. In one to three sentences, how did the study get at this hypothesis/idea? Stated differently, what was the experimental design that allowed the idea to be examined? What is the method that is used in the study? . 4. Select a minimum of one figure or table from the paper, and insert a copy of the figure/table into this report. For this figure/table, describe: 1) why the researchers performed the experiment, 2) the experimental method used to collect the data (may be more detailed than above answer), and 3) an interpretation of the results. 5. Do the results of the paper support the hypothesis/idea? Why or why not?

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