ARTICLE IN PRESS. Animal Behaviour

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1 Animal Behaviour xxx (2009) 1 8 Contents lists available at ScienceDirect Animal Behaviour journal homepage: On the function of an enigmatic ornament: wattles increase the conspicuousness of visual displays in male fowl Carolynn L. Smith *, Daniel A. Van Dyk, Phillip W. Taylor, Christopher S. Evans Centre for the Integrative Study of Animal Behaviour, Department of Brain, Behaviour and Evolution, Macquarie University article info Article history: Received 8 June 2009 Initial acceptance 15 July 2009 Final acceptance 23 July 2009 Available online xxx MS. number: A Keywords: female choice Gallus gallus junglefowl multimodal communication multiple ornament sensory ecology sexual selection signal design tidbitting visual display Males of many species perform elaborate displays in which multiple ornaments feature prominently. However, female preferences often depend upon both display movements and a subset of the ornaments. This response selectivity means that female choice cannot explain the function of nonpreferred ornaments. These structures may instead have an ancillary function (e.g. enhancing signal efficacy or modifying information content). Male junglefowl, Gallus gallus, possess multiple fleshy ornaments, which feature prominently during food-related displays (tidbitting). There is strong evidence for female choice based on display frequency and comb characteristics, but little evidence for choice based on wattles. Wattles are thin, elastic structures that hang loosely from a male s lower mandibles and vary in size over a male s lifetime. These structures swing rapidly during tidbitting, potentially increasing the area around the head and increasing image motion. Males also tidbit more vigorously with highly preferred food, increasing wattle displacement and thereby potentially affecting information content. We tested the prediction that wattles enhance signal efficacy and information content by conducting high-definition playbacks, using three-dimensional animations of tidbitting males with differing wattle properties. Results revealed that the food-searching response of receivers was robust to changes in wattle size and motion. Increased wattle displacement did not decrease orienting latency or increase food-searching duration, which suggests that wattles do not contribute significantly to information content. However, apparent wattle size significantly decreased orienting latency, demonstrating that wattles increase the conspicuousness of the tidbitting signal. These results suggest that wattles are maintained because they enhance signal efficacy. Ó 2009 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. Many animals perform complex visual signals in which elaborate ornaments are displayed prominently (e.g. peacock train fanning: Petrie et al. 1991; frigatebird gular sac inflation: Madsen et al. 2004; wolf spider courtship: Uetz & Roberts 2002). These visual displays and the structural design of the ornaments interact with the receiver s sensory and perceptual abilities and the signalling environment to determine signal efficacy (e.g. conspicuousness and discriminability from other similar behaviours; Guilford & Dawkins 1991, 1993; Endler 1992; Rowe 1999). In addition, the properties of display (e.g. speed, duration, orientation) and the characteristics of the ornaments (e.g. size, colour, movement) can contribute to the signal information content (Zahavi 1979; Hasson 1989; Candolin 2003; Galván 2008). The elongate tail of the male red-collared widowbird, Euplectes ardens, demonstrates how display behaviour increases signal detectability * Correspondence: C. L. Smith, Department of Brain, Behaviour and Evolution, Macquarie University, Sydney, NSW, Australia address: kls@galliform.bhs.mq.edu.au (C.L. Smith). and facilitates assessment of the signal content. Female redcollared widowbirds prefer males with long tails. Males perform courtship flights across open savannah during which the tail is spread laterally and ventrally. Longer tails increase the display conspicuousness (Pryke et al. 2001), and the position of the tail during flight may facilitate female assessment of length (Pryke & Andersson 2005). However, many species possess more than one prominent ornament. In particular, leking and polygynous birds often have both colourful feathers and fleshy structures, which are presented synchronously during visual displays. There are many examples in which female preference depends upon display properties, together with a subset of these ornaments (e.g. peacock: Loyau et al. 2005; frigatebirds: Madsen et al. 2004; fowl: Zuk et al. 1995). This response selectivity means that female choice cannot be invoked to directly explain the function of the other ornaments. One possible explanation is that the nonpreferred ornaments do not currently signal male condition (i.e. that these are unreliable signals; sensu Møller & Pomiankowski 1993). These structures may instead have an ancillary function such as enhancing or increasing /$38.00 Ó 2009 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. doi: /j.anbehav

2 2 C.L. Smith et al. / Animal Behaviour xxx (2009) 1 8 signal efficacy or modifying the information content (e.g. by revealing signaller motivation; Morton 1977). Male junglefowl, Gallus gallus, possess multiple fleshy ornaments on the head (Darwin 1871), which feature prominently during food-related displays (Stokes 1971). The comb is a single, medially located, turgid, red structure on top of the head. In contrast, the wattles are thin, paired, fleshy red protuberances that hang loosely from the lower the mandibles (Fig. 1a). Previous research has demonstrated female choice based on display performance (Pizzari 2003) and a subset of these ornaments (reviewed in Parker & Ligon 2003). Female preference depends upon comb characteristics (e.g. size, colour, hue; Parker & Ligon 2003), but despite a close correlation between comb and wattle properties, there is little evidence that wattles play any role in female mate choice (Zuk et al. 1990; Ligon et al. 1998; but see Zuk et al. 1992). The function of the wattles hence remains enigmatic. Pizzari (2003) demonstrated female preference for males based on frequency of food-related displays, known as tidbitting (Davis & Domm 1943). This is a multimodal signal, typically composed of pulsatile vocalizations (food calls) and rhythmically repeated movements of the head and neck, including picking up and dropping of a food item (Davis & Domm 1943; Stokes & Williams 1972; Evans & Marler 1994). During the tidbitting display, the wattles (Fig. 1b d) swing rapidly with each movement of the head. Females respond to tidbitting by approaching and searching for food near the male (Stokes & Williams 1972; Marler et al. 1986a, b; Gyger & Marler 1988). Previous research (Graves et al. 1985) revealed that female proximity to a male is correlated with the male s mating success and that females remain close to tidbitting males longer than they do nonsignalling males (Smith & Evans 2009). In this hierarchical social system (Collias & Joos 1953), 30% of subordinate displays involve suppression of the call and are composed of the movements alone (C. L. Smith, unpublished data). Playback experiments revealed that the sensory modalities are redundant (sensu Partan & Marler 2005); these purely visual displays are as effective Figure 1. Wattle movement during the tidbitting display. Wattles (a) hang loosely from the male s mandibles when the head is stationary, (b) lag behind the horizontal movement of the head during the twitch motor pattern, (c) tend to swing forward and together during shorter vertical short bob movements, and (d) separate and swing outwards and upwards during the downward phase of the longer vertical long bob motions, occasionally swinging far enough to slap the side of the male s head and then swing back together during the upward phase. as the vocalization alone or the multimodal display in evoking food searching by the hens (Smith & Evans 2008). The tidbitting display is composed of three distinct head and neck movements: twitch : a rapid horizontal side-to-side motion with the neck held fully upright; short bob : an abrupt vertical plunge from the upright position to a point halfway above the ground, returning to the upright position; and long bob : vertical movement of the head, plunging through the full arc towards the ground and ending in the upright pose (see Fig. 1 in Smith & Evans 2009). Each of these motor patterns imparts momentum into the wattle, causing it to swing (Fig. 1a). During a twitch, the wattle lags behind the head at the start of the movement and then continues further in the direction of the movement after the head has reached its furthest lateral displacement (Fig. 1b). During shorter vertical movements, the wattles swing forward and together (Fig. 1c). However, during longer vertical movements, they separate and swing outwards and upwards during the downward phase of the motion, occasionally swinging far enough to slap the side of the male s head (Fig. 1d), and then swing back together during the upward phase. Wattle size is testosterone dependent (Domm 1939; Ligon et al. 1990), and loss of social status results in a reduction in wattle size (Ligon et al. 1990). Males perform these movements at a significantly higher rate during tidbitting than during self-feeding (C. L. Smith & C. S. Evans, unpublished data), suggesting that this increased frequency of motion reflects selection for signal function. Males show a higher rate of food calling (Marler et al. 1986a) and movements (Stokes 1971) when presented with a highly preferred food item. Faster head and neck movements increase the amplitude of wattle motion, suggesting that these elastic structures may facilitate detection of the male display by females, as well as providing an indication of the quality of the food item. The physical characteristics of the wattle, its pronounced movement during tidbitting and the likelihood that hens will experience the unimodal form of the display at a reasonable frequency, when considered together, generate the prediction that these structures have a hitherto unsuspected function, which is to enhance efficacy and/or increase the information content of the tidbitting signal. Here we report an experimental test of the wattles effect on signal efficacy and information content of the tidbitting display. We created high-resolution three-dimensional animations that precisely reproduced the movement of live males performing the tidbitting display and manipulated the properties of the wattles to create four treatments: wattleless (structure absent), blade (structure rigid), normal (displacement matched to donor male) and extrafloppy (displacement exaggerated). We then conducted playback experiments using high-definition plasma screens, a method that is well established in this system (Evans & Marler 1991; Evans et al. 1993a, b; McQuoid & Galef 1993; Clark & Jones 2001; Smith & Evans 2008, 2009) to test female responses. We tested for differences in the latency to orient, the latency to food-search after orienting to the signal and the overall food-search duration based variation in the presence, speed and movement of the wattles. The efficacy hypothesis predicts reduced latency to orient in treatments containing wattles and/or wattle movements, and the information content hypothesis predicts that hens will begin to food-search sooner and continue to food-search for longer based on the presence and movement of the wattles. METHODS Subjects We used 26 golden Sebright bantam females for this study; 24 were used as test subjects and two as audience hens. All hens

3 C.L. Smith et al. / Animal Behaviour xxx (2009) were housed in pairs with ad libitum access to food (Gordon Specialty Feeds laying ration, Sydney, Australia) and water in m cages in a climate-controlled room maintained at 22 C on a 12:12 h light:dark cycle. The behaviour of Sebrights closely resembles that of the ancestral form, the red junglefowl, Gallus gallus (Collias & Joos 1953; Collias 1987; Andersson et al. 2001; Schütz & Jensen 2001), from which all domesticated strains have been derived (Fumihito et al. 1994, 1996). In particular, Sebrights have not been subjected to artificial selection for rapid growth or increased egg production. Ethical Note The protocols used in this experiment conformed to the Animal Behavior Society s Guidelines for the Use of Animals in Research and were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (NHMRC 1997). All procedures were approved under Macquarie University AEC protocol 2006/025. Experimental Design We wanted to test the contribution of the wattles to the efficacy of the tidbitting display, including whether or not the presence of these structures and their movements enhanced conspicuousness, signal recognition and information content. This required us systematically to modify the presence of the wattle and its elastic properties, as revealed by their deformation during head movements, while maintaining all other characteristics of the tidbitting signal (e.g. number and types of movement) and the background constant. To accomplish this, we created an animated threedimensional model of a male and modified the wattles while holding all other aspects of morphology and behaviour constant. Fowl live in stable social groups (Collias & Collias 1996). Females hence reliably experience the same male tidbitting over the course of several seasons (Collias et al. 1966) and are known to respond to the majority of these displays (Gyger & Marler 1988). However, a male s wattles vary in size over the course of his life (Zuk & Johnsen 1998). Females thus experience the same male tidbitting with potentially larger or smaller wattles than during previous encounters. This generates the prediction that recognition of the tidbitting display should not be wholly dependent on wattles exceeding a specific threshold size. We tested this prediction by generating a male that performed normal tidbitting movements but lacked these structures (hereafter referred to as wattleless ). Wattles also have two obvious effects on the tidbitting display; they increase the overall apparent size of the red region around the head, and they generate additional image motion because of their flexible nature. Larger moving objects stimulate larger regions of the retina, thereby facilitating detection of an object (Rowland 1989). Similarly, image motion reliably enhances conspicuousness (Fleishman 1988; Eckert & Zeil 2001). To assess the contribution of the apparent size and apparent motion to signal efficacy, we also modified the movement and elasticity of the wattle structure. The increase in the area of the head region attributable to wattles may facilitate detection of the tidbitting movements regardless of the movement of the wattles themselves. To test whether the apparent size altered the conspicuousness of the display, we created a male with rigid wattles (hereafter referred to as blade ), which mimicked the rigidity of the comb (Ligon et al. 1998). To test the effect of the apparent motion of the wattles, we increased the apparent elasticity of the wattles by exaggerating the swing imparted by each movement of the head (hereafter referred to as extrafloppy ). A male with normal wattles was generated to provide a comparison for females responses (hereafter referred to as normal ). We note that the contribution of apparent size and image motion to signal efficacy generates opposing predictions. The blade wattles do not deform; therefore, the full area of the wattles is fully visible to the hen throughout the display. In contrast, the more flexible the structure, the more often it deforms in such a way as to appear smaller. If the principal contribution to efficacy is from the effect of apparent size, we predicted a shorter latency to orient to the stimuli with increased apparent size during the display (i.e. from shortest to longest latency: blade, normal, extrafloppy, wattleless ). If image motion is the key attribute, we predicted that the stimuli with the greatest apparent motion should generate the shortest latency (e.g. extrafloppy, normal, blade, wattleless ). A complementary analysis was concerned with information content. Males tidbit more vigorously when highly preferred food is discovered (Stokes 1971). There may therefore be a predictive relationship between food quality and the degree of wattle movement. This generates the prediction that food-search duration should increase as a function of the elasticity of the wattles. When the movement of the wattles is artificially enhanced ( extrafloppy ), this should have the same effect as faster head movements on foodsearching duration (e.g. from longest to shortest duration: extrafloppy, normal, blade, wattleless ). Animations that precisely match the movements of live animals allow researchers to modify specific visual attributes while holding all other morphological and behavioural cues constant (Rosenthal & Evans 1998; Watanabe & Troje 2006). This technique has been used successfully to elicit natural responses in a wide array of taxa, including birds (Watanabe & Troje 2006), lizards (Peters & Evans 2003), fish (Rosenthal & Evans 1998) and spiders (Uetz & Roberts 2002). Animation Methods We obtained a three-dimensional computer model of a red junglefowl and skin texture from a commercial supplier (Turbo Squid, New Orleans, LA, U.S.A.). We imported the model into an animation program (LightWave 3D v8.3, NewTek Inc., San Antonio, TX, U.S.A.) running on a Power Mac G5 (Apple Computer, Inc.). We modified the model so that it more closely matched the morphology of the exemplar roosters. We did not alter the model s plumage because previous research (Ligon & Zwartjes 1995) determined that females do not show a preference for males with plumage similar to their own. We defined the range of motion by creating virtual bones in the head, neck and wattles (see Peters & Evans 2003, 2007; Van Dyk & Evans 2008). We animated the tidbitting displays using rotoscoping, a technique that involves matching the configuration of a virtual object to sequential frames of a real one (Peters & Evans 2003, 2007; Van Dyk & Evans 2008). For the video sequences, we used archived highdefinition recordings of two tidbitting males that had been filmed in a sound-attenuating chamber (for details of the filming set-up see Smith & Evans 2008). We exported the first 10 s of each male s tidbitting display as resolution JPEG still images. In LightWave 3D, we set the position of the virtual camera to replicate the focal length and orientation of the original video camera and then, through an iterative process, imported each of the 256 frames and manipulated the model rooster s skeleton so that the position of each body part matched the corresponding body part of the videoed rooster in every frame. The result was an animated sequence that precisely replicated the motion characteristics of the original video sequence of display by a live male. This allowed manipulation of structure and movement characteristics in a way that would not be possible with digital video recordings. Tidbitting displays are typically viewed by hens against a complex vegetation background. To emulate this, we recorded

4 4 C.L. Smith et al. / Animal Behaviour xxx (2009) 1 8 digital video of an outside garden on a sunny day with short grass in the foreground and large green ferns in the background. A single interlaced frame was set as background in LightWave 3D so that the animated rooster appeared to be standing on the grass in front of the ferns (see Supplementary Material normal.mp4 ). We used the animated sequence to create four treatments from each exemplar male. The normal treatment was built from unmodified display sequences (see Supplementary Material normal.mp4 ; Fig. 2a). To create the wattleless sequence, we removed the wattles from the rooster model (see Supplementary Material wattleless.mp4 ; Fig. 2b). The blade sequence was created by fixing the virtual bones controlling the wattle s motion so that the position of this structure relative to the head did not change during the display sequence (see Supplementary Material blade.mp4 ; Fig. 2c). The extrafloppy treatment required a consistently exaggerated motion (see Supplementary Material extrafloppy.mp4 ; Fig. 2d). We achieved this by selecting the first two virtual bones in the middle of each wattle (i.e. those closest to the head) and increasing their displacement. We measured the difference in bone position in the X, Y and Z axes from one frame to the next and then multiplied this by a factor of 2.5, repeating the process throughout the sequence. On a few occasions, this formula resulted in the wattles moving in impossible ways such as entering the beak or neck. In these instances, we constrained movement so that it remained physically possible. Finally, we created a control motionless male animation in which the first frame was repeated for the entire sequence. The final playback stimuli were each 2 min long. This allowed the hen sufficient time to respond and is well within the normal duration of tidbitting with a highly preferred food item (e.g. mealworms; C. L. Smith, unpublished data). Since it would have been impractical to rotoscope a sequence of this length (3072 frames by two males by four treatments), each requiring individual position calculation and adjustment of the bones in three axes, we looped the 10 s animated sequence. To avoid motion artefacts, Figure 2. Example of the difference between the wattle movement in the four treatments. Frame (no. 106) from the each of the four treatments. Head, neck and body movements were identical across all four treatments. Wattles were modified as follows (a) normal : wattle movement identical to video exemplar males, (b) wattleless : removed, (c) blade : rigid structure, movement in line with the lower mandible, (d) extrafloppy : 2.5 times the distance moved in the normal treatment. we added a six-frame transition at the end of each 10 s animated sequence in which the model returned to its original position. Prior to rendering, the virtual camera in LightWave 3D was positioned so that the animated rooster appeared side-on. We chose the lateral orientation because observational studies of natural interactions have shown that hens preferentially approach a tidbitting male from the side (C. L. Smith, unpublished data). We exported each animation and the background sequence without a male as sequential JPEGS and then used QuickTime (Apple Computer, Inc.) to compile the images into a video clip. From the video clips, we created 11 playback sequences (two exemplars by four treatment types each, a motionless male sequence of each exemplar, and a background sequence), each of 12 min total duration. The first 10 min of every playback was the background vegetation sequence, with no male present. The following 2 min were one of the four treatment types or the motionless male. Playback Procedure During playbacks, we used audience hens to facilitate the acclimation of the female being tested (focal hen) and to create a more natural setting since fowl are group-living animals. The same two audience females were present during each trial. To ensure that they could not influence the response of the focal hen, the apparatus was designed so that the audience hens could not see the playback stimuli. Each focal hen was first randomly assigned to an exemplar male and then to a unique randomized sequence of the four test stimuli generated from that male. We conducted video playback experiments in a m (width length) outdoor field cage. This was a steel-framed structure covered with several layers of shade cloth, which eliminated direct sunlight and prevented the focal hen from experiencing external visual distractions. The test apparatus consisted of three cloth covered cages (each m) housing the focal and audience hens and two Mac mini computers (Apple Computer, Inc.), which controlled playbacks on two 106 cm Sony high-definition flat panel plasma displays (Sony PFM-42X1, resolution pixels). The hens responses were digitally recorded from two angles to ensure coverage of the entire test area. We used QuickTime to record the trials in real time in native resolution onto two Mac mini computers using a Sony high-definition video camera (HDR-HC7) and a DV Canon HandyCam (830i) with Raynox wide angle lens (HD-5000PRO). We aligned the three cages along the 6 m wall so that the middle of the centre cage was 3 m from either end. During testing, we placed the focal hen in the centre cage and an audience hen in each of the outer cages. The audience hen cages were covered on three sides in opaque cloth, preventing the audience hens from seeing into the test chamber but allowing them to visually and vocally interact with the each other and with the focal hen. The front side of the centre cage consisted of two side-hinged doors that were latched in the centre and could be remotely released by an external observer. We situated the plasma displays in the far corners on the opposite wall from the cages and oriented them at a 45-degree angle to the front of the centre cage. The plasma displays were enclosed in matte green wire mesh, which prevented hens from approaching closer than 30 cm, the maximum distance at which females are able to recognize individual conspecifics (Dawkins 1995). This spatial separation was also sufficient to prevent hens from resolving individual pixels on the displays. We also surrounded the displays with ferns similar to those in the video background to camouflage the housings and increase the realism of the scene. We acclimated the audience and focal hens to the test chamber setup for 6 days prior to testing. The procedure was identical to the test

5 C.L. Smith et al. / Animal Behaviour xxx (2009) days,withtheexceptionthatbothplasmascreensshowedthefern background with no male present for the entire acclimation period. This process ensured that the remote door opening did not startle the focal hen so that she walked freely throughout the test chamber. At the beginning of the test day, we placed the two audience hens in their respective cages. At the start of each 12 min trial, we placed the focal hen in the centre cage with the door closed and then activated the playbacks on the plasma displays. The cage door remained closed for 10 min, which allowed the hen sufficient time to settle down after handling. After 10 min of playback, the treatment stimulus appeared on one of the plasma screens and the motionless male appeared on the other. Synchronous with this, the cage door was opened using remote control, allowing the focal hen to see and approach the plasma screens. Each hen was tested at the same time each day (i.e. intertrial interval was 24 h). We alternated assignment of the treatment and motionless males between the two screens to prevent hens from anticipating the location of the displaying male. Behaviour during the 2 min treatment period was scored using JWatcher Video 1.0 (Blumstein et al. 2006), which reads the timecode of the video file to permit single-frame resolution (40 ms in the PAL standard). We measured the latency to orient towards the treatment playback stimulus, latency to onset of food searching and duration of food searching. Fowl have limited movement of the eye in the socket and therefore use jerky head movements to bring objects into focus on the fovea of either eye. They consequently perform a distinctive, high-amplitude head swing when they first see a conspecific (Dawkins 2002). Orientation latency was measured as the time from the door opening to the first time the female s head swung towards the displaying male. The majority of these head movements were accompanied by stretching of the neck towards the plasma screen with the displaying animated male, a response that was never observed to the plasma screen showing the motionless male. Latency to onset of food searching was measured from time the hen oriented towards the treatment stimuli to the first time she fixated on the substrate in the characteristic food-searching posture (Evans & Evans 1999, 2007). Food-searching duration was the total time the female spent fixating on the substrate from the onset to the end of the 2 min trial. Data were analysed with a linear mixed model (SPSS for Macintosh) using treatment type, male exemplar, test session (morning or evening) and trial day number as fixed effects. Hen identity was included in the model as a random factor. We treated male exemplar as a fixed effect to test whether female responses were robust to differences in display composition. Tidbitting displays are highly variable, with no indication of temporal stereotypy (Smith & Evans 2009). Both 10 s rotoscoped displays contained one long bob, but the ratio of twitches to short bobs during the rest of the display differed between the two exemplars. Latency data sets were normalized using log transformation prior to analysis. When treatment type was significant, further pairwise comparisons of the means, adjusted for all variables included in the model, were tested for significance. We used the false discovery rate (FDR) method to determine significant differences between means (Benjamini & Hochberg 1995). This test balances the likelihood of type I versus type II errors when making multiple pairwise comparisons and has been demonstrated to reduce the number of type II errors compared to other tests that similarly control the familywise error rate (Verhoeven et al. 2005). RESULTS Orientation Latency Analysis of the latency to orient towards the tidbitting animation revealed that treatment type (F 3,183 ¼ 7.270, P ¼ ) and trial day (F 1,183 ¼ 3.690, P ¼ 0.048) were significant. Exemplar and test session were not significant (F 1,183 ¼ 0.001, P ¼ and F 1,183 ¼ 0.534, P ¼ 0.466, respectively). Pairwise comparison of the adjusted means of the four treatment types revealed that females took significantly longer to orient towards the wattleless treatment than to any of the other treatments (P < 0.05, adjusted using FDR). There was no significant difference in the latency to orient to the normal treatment compared to the blade or extrafloppy treatment, however, blade had a significantly shorter latency than extrafloppy (P < 0.05, adjusted using FDR; Fig. 3). Latency to Onset of Food Searching We also analysed the hen s latency to onset of food searching after orienting to the test stimuli. We used this as a measure of the effect of the wattle on signal recognition. Both treatment type (F 3,159 ¼ 2.780, P ¼ 0.043) and trial day (F 1,159 ¼ 30.06, P < 0.001) were significant. Exemplar and test session were not significant (F 1,21 ¼ 0.549, P ¼ and F 1,21 ¼ 3.733, P ¼ 0.067, respectively). We then conducted pairwise comparisons of the adjusted means of the four treatment types. Using FDR to adjust the pairwise comparison, we found no significant difference in the latency to onset of food searching between any of the four treatment types (P > 0.05, adjusted using FDR; Fig. 4). Food-searching Duration We measured the duration of the hens food searching in response to the four treatments and found no significant differences to the treatment type (F 3,162 ¼ 0.514, P ¼ 0.673), exemplar (F 1,21 ¼ 0.910, P ¼ 0.766), test session (F 1,21 ¼ 1.623, P ¼ 0.217) or trial day factors (F 1,162 ¼ 0.488, P ¼ 0.486; Fig. 5). DISCUSSION The results of the playbacks revealed that females oriented sooner to the stimuli with the larger overall head region ( blade and normal ) than to those with faster-moving wattles ( extrafloppy ) or to the stimuli that lacked these structures entirely ( wattleless Fig. 3). In the blade stimuli, the wattles mimicked the Latency (frames; log adjusted) a b Wattleless Blade Normal Extrafloppy Treatment Figure 3. Latency to orient to tidbitting signal. Adjusted mean SE time to hens first orientation to tidbitting stimuli (log adjusted). Orientation was measured as the time from the door opening, which allowed the hen to view the stimuli, to the first time the female s head swung towards the tidbitting treatment. Different letters indicate significance (P < 0.05, FDR adjusted). bc c

6 6 C.L. Smith et al. / Animal Behaviour xxx (2009) NS 50 NS Latency (frames; log adjusted) Food searching duration (s) Wattleless Blade Normal Extrafloppy Treatment 0 Wattleless Blade Normal Extrafloppy Treatment Figure 4. Latency to onset of food searching. Adjusted mean SE latency to onset of food searching (log adjusted), as measured by the first time of close binocular fixation on the substrate after orienting to the tidbitting stimulus. Figure 5. Food-searching duration. Adjusted mean SE time that hens spent food searching after orienting to tidbitting stimulus. Food searching was measured as time spent in close binocular fixation on the substrate. rigid structure of the comb and therefore presented the maximum area throughout the entire display. In contrast, in the extrafloppy condition, the wattle often swung up beside the male s head during fast movements, potentially decreasing the apparent size of the structure. This suggests that the apparent size of the wattle contributes more to the conspicuousness of the tidbitting display than does the apparent motion of the wattle. Based on many of fowl s social and life history parameters (see Introduction and Methods), we predicted that the female s ability to recognize the tidbitting signal should be robust to changes in wattle elasticity. Playback results confirmed our prediction regarding signal recognition; females latency to onset of food searching (Fig. 4) and duration of food searching were unaffected by the presence or movement of the wattles (Fig. 5). We conclude that once the receiver s attention is engaged, recognition of the tidbitting signal is based on other characteristics, such as the rate or types of movements of the head and neck. In addition, these results suggest that female s response to tidbitting is robust to differences in the movement composition of the display. There were no significant differences in the females latency to orient, latency to food-search or duration of food-searching response between the two playback exemplars, even though these were quite different. This permissive response probably reflects the temporal constraint imposed by female female competition. Only the first hen to approach a tidbitting male receives food (Stokes & Wiliams 1971), which mandates rapid assessment of signal structure. Furthermore, the movement characteristics of the wattle do not appear to convey additional information regarding the palatability of the food item; hens did not begin to search for food sooner (Fig. 4) or longer (Fig. 5) in response to the extrafloppy treatment compared to normal. It is possible that the increase in wattle motion was not sufficient to elicit a response, or perhaps more likely that the overall movement of the head and neck are more visually salient than the movements of the comparatively small wattles. The results of this experiment also raise an interesting question regarding the selection pressures that maintain condition-dependent traits in fowl. There is strong evidence to suggest that female fowl select males based on the properties of the comb (for overview see Parker & Ligon 2003), the size of which is highly correlated with wattle size (Ligon et al. 1998). Both of these fleshy ornaments are testosterone-dependent traits (Zuk et al. 1995) as well as indicators of immunocompetence (Zuk et al. 1995; Zuk & Johnsen 1998; Verhulst et al. 1999). Therefore, we might expect that females would show an equally strong preference for wattle characteristics as for combs. However, the majority of previous research (Zuk et al. 1990; Ligon et al. 1998; except see Zuk et al. 1992) has found no evidence of female preference for any characteristic of the wattles (e.g. size, colour or symmetry), despite its potential as an honest signal (Zuk 1991; Zuk & Johnsen 1998). In species such as fowl that possess multiple ornaments, only a subset of which function in mate choice, it has been hypothesized that the nonpreferred ornaments may be unreliable signals, in the sense that they do not currently indicate male quality (Møller & Pomiankowski 1993). These ornaments may have arisen because of a pre-existing female bias that was unconnected to mate choice (Arak & Enquist 1993), although, they may be maintained if they increase the efficacy of other signals (Endler & Basolo 1998). Gallus gallus chicks have an innate preference for the colour red (Salzena et al. 1971; van Kampen & de Vos 1991), which persists into adulthood, with females preferring males with redder ornaments (Zuk et al. 1990). It is possible that the colour of the comb and wattles exploited this well-documented sensory bias (Jansson & Enquist 2003, 2005). However, there are substantial differences between the internal structure of the comb and wattles such that combs may provide a more reliable indication of condition than wattles (Ligon et al. 1998). The results of our playback experiment demonstrate that wattles increase the conspicuousness of the tidbitting signal, as measured by orienting latency. This is consistent with the idea that the structures are maintained because they enhance signal efficacy. Acknowledgments We thank R. Miller and C. Jude for bird care, and R. Marshall for veterinary support. We also thank A. Taylor for assistance with the statistical analysis. This research supported by a grant to C. S. E. and P. W. T. from the Australian Research Council and funding to C. L. S. from Macquarie University. Supplementary Material Supplementary Material for this article may be found in the online version at doi: /j.anbehav

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