Egg incubation temperature affects male reproductive ...

Behav Ecol Sociobiol DOI 10.1007/s00265-009-0897-0

ORIGINAL PAPER

Egg incubation temperature affects male reproductive success but not display behaviors in lizards Daniel A. Warner & Kevin L. Woo & Daniel A. Van Dyk & Christopher S. Evans & Richard Shine

Received: 31 July 2009 / Revised: 4 December 2009 / Accepted: 5 December 2009 # Springer-Verlag 2009

Abstract The complex ritualized displays of males in many territorial species suggest that selection has shaped male behaviors in ways that affect fitness. In this study, we evaluated the link between display behavior during male– male interactions and reproductive success in the Australian jacky dragon (Amphibolurus muricatus), a lizard species that uses a complex series of movement patterns for communication. We quantified variation in male display behaviors by using video playback experiments in the laboratory, and subsequently assessed variation in male reproductive success by paternity analyses of offspring. Because the lizards used in this study came from eggs incubated under three thermal environments, we also could evaluate the impact of developmental temperature on adult behavior and reproductive success. Incubation temperature had a strong effect on male reproductive success; males Communicated by S. Downes D. A. Warner : R. Shine School of Biological Sciences, University of Sydney, Sydney, NSW 2006, Australia K. L. Woo : D. A. Van Dyk : C. S. Evans Centre for the Integrative Study of Animal Behaviour, Macquarie University, Sydney, NSW 2109, Australia Present Address: D. A. Warner (*) Department of Ecology, Evolution and Organismal Biology, Iowa State University, Ames, IA 50011, USA e-mail: [email protected] Present Address: K. L. Woo Department of Biological Sciences, University of Central Florida, Orlando, FL 32816, USA

produced under intermediate temperatures sired more offspring than those produced under extreme developmental temperatures. However, incubation temperature did not affect male display behavior, nor was male behavior associated with reproductive success. Our findings do not support the common assumption that display behaviors used during male–male interactions affect reproductive success. Keywords Aggressive behavior . Amphibolurus muricatus . Selection gradient . Submissive behavior . Temperature-dependent sex determination . Video playback experiment

Introduction Identifying the sources of variation in fitness is a major goal of evolutionary biology (Arnold 1983). By understanding how phenotypic traits influence individual fitness, we can gain insights into how traits evolve in response to selection. In particular, traits that are associated with territory defense and mate acquisition are presumably under strong selection because they have direct consequences on mating success and, hence, fitness (Brown and Orians 1970; Pemberton et al. 1992). Indeed, selection has generated an impressive diversity of morphological and behavioral traits that enhance an individual’s ability to defend territories and acquire mates in a diversity of taxa, including invertebrates (Cade and Cade 1992; Murai and Backwell 2006), fish (Yabuta 2000), reptiles (Lovern and Jenssen 2003), birds (Rowe 1999), and mammals (Pemberton et al. 1992). Many animals exhibit highly complex and ritualized display behavior, especially by males of territorial species (e.g., Fox 1983; Latruff et al. 1999; Rowe 1999; Murai and Backwell 2006; Peters and Ord 2003; Fitze et al. 2008). For

Behav Ecol Sociobiol

example, male fiddler crabs perform complex claw-waving behaviors to attract mates (Latruff et al. 1999; Murai and Backwell 2006); similar arm-waving behaviors have been described in some frogs (Grafe and Wanger 2007). Analogously, male Anolis lizards use a series of head bobbing and push-up displays to communicate with conspecifics during courtship and territorial interactions (e.g., Orrell and Jenssen 2003). This complexity of male display behavior suggests that selection has likely shaped the form of displays and that behavioral variation among males within a population should influence variation in mating success. However, although male display behaviors are presumably important for fitness, few studies have tested this prediction by evaluating links between behavior and siring success (e.g., Durães et al. 2009; Ryder et al. 2009). Hence, studies that directly evaluate the relationship between behavior and reproductive success will provide important contributions to our understanding of the link between behavioral ecology and microevolution. Many squamate reptiles are excellent models for evaluating the role of display behaviors in generating variation in fitness. In many lizard species, for example, adult males exhibit complex stereotyped display behaviors that are easily quantified (Carpenter and Ferguson 1977; Lovern and Jenssen 2003; Peters and Ord 2003). These behaviors often are associated with territory defense and mate acquisition and, hence, have important implications for individual fitness. Although relationships between fitness and morphology/performance have been evaluated (e.g., Gullberg et al. 1997; Lappin and Husak 2005; Husak et al. 2006), relationships between display behavior and fitness in lizards have not been explored. In the present study, we evaluate display behaviors and reproductive success in male lizards to understand the role of these behaviors in generating variation in fitness. Because incubation temperature can affect behavioral phenotypes in lizards (Gutzke and Crews 1988; Flores et al. 1994; Downes and Shine 1999), we attempted to amplify variance in male behavior by incubating eggs across a range of temperatures, as would occur under natural conditions. The resultant offspring were then raised under standardized conditions to minimize the effects of juvenile experience on adult behavior. After all lizards reached adulthood (3 years after hatching), we employed a video playback experiment (Clark and Uetz 1990; Evans and Marler 1991) to evaluate the link between behavioral performance and reproductive success (assessed through siring success using microsatellite genetic markers). This video playback technique allows presentation of a standard simulated opponent and reliably evokes display responses in our study species (Ord et al. 2002; Peters and Evans 2003a, b; Van Dyk et al. 2007). Additionally, our design enabled us to evaluate how developmental temperature may impact

both display behavior and reproductive success. We predict that males that consistently perform aggressive displays will have greater reproductive success than individuals that perform submissive displays.

Materials and methods Study species The jacky dragon (Amphibolurus muricatus) is an agamid lizard abundant in coastal heathland habitat in southeastern Australia. Jacky dragons produce highly stereotyped displays during social interactions, consisting of an introductory tailflick followed by a backward and forward arm wave, and then a pushup-body-rock. In jacky dragons, these display behaviors are associated with territory defense during male– male interactions (Peters and Ord 2003). As in most lizards, such display behaviors are involved in communicating territory ownership, creating dominance hierarchies, and may be used in courtship and, hence, likely play an important role in mate acquisition and overall reproductive fitness (Baird et al. 2003). However, the relationship between display behavior and reproductive success has not been evaluated previously in this species. Our previous studies of jacky dragons demonstrate that incubation temperature affects several fitness-relevant traits of hatchlings, such as body size and growth (Warner and Shine 2005, 2008a). In addition, jacky dragons exhibit temperature-dependent sex determination (TSD), whereby both sexes are produced at intermediate incubation temperatures and only females are produced at either extreme (Harlow and Taylor 2000). Our experimental work has shown that male reproductive success is optimized by incubation temperatures that naturally produce males (Warner and Shine 2008a) but the performance mechanism responsible for this effect remains unknown. Thus, the current study not only allows us to evaluate the critical link between male display behavior and fitness but also enables us to determine if male–male display behaviors mediate the relationship between incubation temperature and male fitness. Source of eggs and incubation experiment The lizards used in this study are a subset of those used in past experiments and, therefore, methodology for egg collection and incubation has been described previously (Warner and Shine 2005, 2008a). Here, we provide only brief details of the source of eggs (and resultant hatchlings) that were used in this study. In spring 2003, gravid A. muricatus were collected from natural areas surrounding Sydney and housed in outdoor enclosures until they


oviposited. Eggs were collected (221 eggs from 41 clutches) and incubated under three temperature regimes (23, 27, and 33°C) with daily fluctuations of ±5°C that mimicked natural nest temperatures (Warner and Shine 2008b). In jacky dragons, cool (30°C) incubation temperatures produce females and intermediate temperatures produce mixed sex ratios (26–30°C; Harlow and Taylor 2000). An aromatase inhibitor was applied to half of the eggs in each treatment to block the conversion of testosterone to estradiol during development, and thereby produce male offspring at female-producing temperatures (Wibbels and Crews 1994). This hormonal manipulation allowed us to decouple the confounded effects of sex and incubation temperature and, hence, evaluate how a range of incubation temperatures affects male behaviors even for individuals that hatched from eggs incubated at naturally female-producing temperatures. Importantly, our sex reversal technique produces males that behave the same as naturally produced males (Balthazart et al. 1994; Wennstrom and Crews 1995; this study). Moreover, our sex reversal technique does not affect male gonadal morphology or reproductive success in our study species (Shine et al. 2007; Warner and Shine 2008a). All hatchlings were uniquely marked by toe clipping, and subsequently released into outdoor field enclosures where they remained until autumn 2007 (lizards were about 3.5 years of age by this time). All toe clips were preserved in 70% ethanol, and DNA from these tissue samples were used for parentage analysis when these animals reproduced (see below). Individuals were released into one of six enclosures (30–32 hatchlings per enclosure), with each treatment and both sexes equally represented among replicate enclosures. Each enclosure (4×8 m) was divided into eight 2×2-m sections. Holes in the inner dividing walls of the enclosures allowed lizards to move freely throughout the entire 4×8-m area, but enabled lizards to move out of visual contact with each other. By housing lizards in these enclosures, the social environment was standardized for each individual lizard when raised to adulthood. All lizards were provided with the same feeding regimes, but competed with each other for food within enclosures (see Warner and Shine (2005) for details of the enclosures and feeding regimes). In late summer 2007, all surviving male lizards were sexually mature and we collected most of the males (n=41 out of 45) to measure display behaviors. Prior to behavioral trials, all lizards were weighed and measured for snout-vent length (SVL), tail length (TL), jaw length, jaw width, and head depth (see Harlow and Taylor (2000) for measures of head dimensions). Lizards were then transported to indoor enclosures where they were allowed to acclimate for one week before testing.

Indoor housing Lizards were individually housed in cages (64 cm wide× 75 cm long×120 cm tall) that contained sand substrate and several branches for basking and perching. Lizards were visually isolated from each other, and maintained on a 14:10 h light/dark cycle. Additional heat lamps above each enclosure were illuminated for only 12 h during the light period. Room temperature was kept at 25±3°C throughout the study. Ultraviolet lamps (300 W Ultra-Vitalux, Osram, NSW, Australia) were illuminated every morning for 15 min. Three times per week, lizards were fed crickets (Achetus domesticus) that were dusted with vitamin supplements (RepCal, Victoria, Australia). Water was available ad libitum. Two cohorts of lizards were subjected to video playback trials. The first cohort of lizards (n=21) was collected from the outdoor enclosures on 22 January 2007. After 2 weeks (1 week of acclimation and 1 week of trials), these lizards were returned to their outdoor enclosures. The indoor cages were cleaned prior to housing the second cohort of 20 lizards on 5 February 2007. Playback stimulus acquisition and video playback trials Protocols for video playback trials have been described in detail previously (Ord et al. 2002; Ord and Evans 2002; Peters and Ord 2003; Van Dyk and Evans 2007). We used archived video footage of jacky dragons from previous studies (Ord et al. 2002; Ord and Evans 2002; Van Dyk and Evans 2007) that showed a life-sized male jacky dragon (45 mm SVL) performing aggressive push-up displays from an artificial perch. The footage was shown to the subject lizards on a high-resolution video monitor placed in front of the cage. We recorded the lizards’ responses using a camera with a wide angle lens connected to a Video Home System recorder. Lizards were shown stimulus clips using a standard digital video playback system (Ord et al. 2002; Ord and Evans 2002; Peters and Evans 2003a; Van Dyk and Evans 2007). Video sequences were stored on a 250 GB LaCie hard drive (Hillsboro, OR, USA) that was connected to an iMac (Apple Computer Inc.) with Final Cut Pro software. The digital video signal was then sent from the iMac to a Canopus® ADVC110 (Grass Valley, Inc., Burbank, CA, USA) for analog conversion and subsequently displayed on a Sony Trinitron monitor (Model No. PVM-14N5A: Sony Corporation, Shinagawa, Tokyo Japan). We recorded all behavioral responses to the stimuli with a Panasonic closedcircuit television camera (WV-CP240/G) that was connected to a color viewfinder (Panasonic TC-1470Y), which was also connected to a videocassette recorder (Sony) for scoring at a later date.


During the week of the trials, each lizard was exposed to one of three 10-min video clips of a displaying male jacky dragon on alternating days (Monday, Wednesday, and Friday). The first 2 min showed an image of the artificial perch. After 2 min, the image showed a male jacky dragon climbing onto the perch, and the remaining 8 min showed sequences of the lizard performing a total of 20 push-upbody rocks, displays typically used in territorial defense or courtship interactions. Trials were repeated three times per individual. The video clips for each trial presented different display sequences, but maintained the same display rate. We randomized the order of video clips shown to each lizard among individual trials, such that no lizard was exposed to the same video clip more than once. All video playback trials took place between 0900 and 1300 h, within the normal activity time for jacky dragons in the field and laboratory (D.A. Warner, personal observation; Ord and Evans 2002). While reviewing the video footage at a later date, we counted the number of times each behavioral response was displayed during the trial periods. The response variables that we measured included counts of a variety of aggressive, submissive, and exploratory behaviors that have been defined in previous studies of jacky dragons (Ord and Evans 2002; Peters and Ord 2003). Aggressive variables included tail-flicks, backward-forward armwaves, pushup-body-rocks, gular extensions, and attacks (Peters and Ord 2003). Submissive variables comprised slow arm waves and slow head bows (Carpenter et al. 1970). Because the function of additional separate movements of more than one body length (i.e., locomotion) and substrate licks is unknown, they were classified as exploratory variables. We suspect that lizards produce these exploratory behaviors to acquire more information (i.e., chemical cues) about their surrounding environment (De Fazio et al. 1977) and they are only elicited in the presence of an opponent during interactions (K.L. Woo, D. A. Van Dyk, personal observation). Male reproductive success Reproductive success of males was measured during the 2006/2007 reproductive season (late September to early January; prior to the video playback trials). Because the males used in the video playback trials were housed with females in their respective outdoor enclosures, individuals could interact and interbreed. During the reproductive season, females were temporarily removed from their enclosures when they showed signs of gravidity, housed individually until they nested, and then returned to their original enclosure. Eggs were collected from the nest site and incubated at a constant 28°C; additional protocols for egg incubation followed those of Warner and Shine (2005).

After these second generation eggs hatched, tissue samples (tail clips) from the resultant hatchlings were used for DNA extraction for paternity analyses. Although reproductive data were collected over three seasons (Warner and Shine 2008a), only data from the 2006/2007 reproductive season were used in the analyses for the current study because this was the same season in which we quantified male behaviors. DNA was isolated from 58 males, including the 41 individuals used in the video playback experiment, and 47 adult females that were still alive at the onset of the breeding season in 2006. We also isolated DNA from all 203 offspring produced in the 2006/2007 breeding season. DNA was isolated from the toe clips and tail clips using Qiagen DNAeasy tissue extraction kit. Samples were then genotyped at the following eight microsatellite loci: AM01, AM16, AM25, AM52b, AM53, CP10, CP11 (Schwartz et al. 2007), and Cpti1C7 (Austin et al. 2006). We performed PCR amplifications using fluorescently labeled primers under the conditions described by Austin et al. (2006) and Schwartz et al. (2007). PCR products were electrophoresed on an ABI 3130x Genetic Analyzer (Applied Biosystems). We scored genotypes by eye with the assistance of Genemapper software (Applied Biosystems). We performed paternity analyses using CERVUS software (Version 3.0.3; Marshall et al. 1998). For each individual enclosure, genotypes from the adults were used to calculate allele frequencies, observed and expected heterozygosities, frequency of null alleles, and the polymorphic information content (a measure of informativeness related to expected heterozygosity) of the loci (Bostein et al. 1980; Hearne et al. 1992). Mother-offspring genotypes were compared for the presence of null alleles. Within these comparisons, loci containing null alleles (AM25) were deleted from the analyses. All males that survived to the onset of the 2006 breeding season were classified as potential fathers. Paternity was initially assigned using two simulation analyses: first using 0 genotyping error rate (complete exclusion), and second using a 0.01 genotyping error rate. The data set was then corrected for any potential null alleles by deleting all homozygous genotypes at locus AM25 from the potential fathers and parentage was assigned a third time using a zero genotyping error rate simulation. Results from all three analyses were manually compared, and the assigned fathers were checked manually across mother-offspring pairs and the clutch mates. Statistical analyses All statistical analyses were performed using SAS software (SAS Institute 1997). Repeatability (r) of behaviors across the three successive trials was evaluated by obtaining the within- and among-individual mean squares from a one-way


analysis of variance for each behavior; these values were then used to calculate r (see Arnold 1994). Because behaviors were repeatable across trials (see Results section), we combined data among behavioral trials by calculating mean response values across trials for each individual; these mean values were then used as our unit of statistical analysis for subsequent analyses. We employed non-parametric analyses to evaluate the effect of incubation temperature on display behavior because assumptions of normality were violated in most cases even after data transformation. In some cases, results from analyses of variance and multiple regression analyses are presented (more details below). Overall, our results did not differ whether parametric or non-parametric tests were used. The effect of incubation temperature on display behavior was evaluated in three ways. First, due to high correlations among several of the behavioral variables, we used principal components analysis to collapse the variables into uncorrelated components. We excluded principal components (PC) that individually explained less that 10% of the variation in the data set (Johnson 1998) leaving four PCs that explained a total of 79.3% of the variation in the data set (Table 1). Aggressive behaviors (i.e., tail flicks, backward-forward arm waves, pushup-body-rock, and gular extension) loaded on the first principal component (PC1). Behaviors associated with exploration (i.e., substrate licks and locomotion) loaded on the second principal component (PC2). Both submissive displays (slow arm wave and slow head bow) and one aggressive display (attack) loaded on the third (PC3), with a submissive display (slow head bow) and aggressive display (attack) on the fourth principal component (PC4). Principal component scores were then used as dependent variables in Kruskal–Wallis tests to evaluate the effect of incubation temperature on behavior. Table 1 Loading values on each principal component (PC) in a principal components analysis

Italics indicates traits represented by each PC

Behavioral trait

By using only males produced at 27°C, we also used Kruskal–Wallis tests to evaluate the effect of the aromatase inhibitor on display behavior (based on PC scores). In a second set of analyses, we classified behaviors as aggressive, submissive, or exploratory as described by previous studies (see above). The sum of each display type was calculated for each individual, and then averaged across the three trials. These mean values were then used as dependent variables in Kruskal–Wallis tests to evaluate the effect of incubation temperature on the number of aggressive, submissive, and exploratory behaviors performed. Our third set of analyses complemented those described above. However, rather than grouping displays into behavioral categories, we used separate Kruskal–Wallis tests to evaluate the effect of incubation temperature on each of the nine displays. In these analyses, the average counts of each display across the three trials were used as dependent variables. By analyzing each display behavior individually, we were able to evaluate if a particular display was important, rather than just broader classes of displays (i.e., aggressive vs. submissive vs. exploratory). The effect of incubation temperature on the latency to respond (number of minutes to first response averaged across trials) was also evaluated with a Kruskal–Wallis test. Multiple regression analysis was used to evaluate relationships between morphology (SVL, TL, and head dimensions) and behavior. Tail length and head size dimensions were corrected for body size by calculating residual scores from the regression of tail length or head size on SVL. Overall statistical patterns did not differ whether behaviors were expressed as averages across trials or as PC scores, thus only analyses based on PC scores are presented here.

PC 2

PC 3

PC 4

0.404 0.529 0.525 0.102 0.445

0.023 −0.071 −0.082 0.222 −0.268

−0.184 −0.012 0.028 −0.575 0.233

0.011 −0.128 −0.151 0.639 0.124

Submissive displays Slow head bow Slow arm wave

−0.018 −0.058

−0.383 0.375

0.476 0.547

0.691 −0.044

Exploratory Substrate lick Locomotion Percentage of variance explained

0.113 0.024 37.6

0.584 0.488 19.3

0.140 0.187 11.8

0.238 0.024 10.5

Aggressive displays Tail flick Back-forward arm wave Pushup-body-rock Attack Gular extension

PC 1


Male reproductive success was evaluated in three different ways. First, we classified individuals as either ‘successful’ or ‘not successful’ at siring offspring, and used a chi-square test to evaluate the effect of incubation temperature on the frequency of successful versus nonsuccessful males. As a second index of reproductive success, we counted the number of clutches in which a given male sired at least one offspring (multiple paternity occurred in 30% of the clutches); this provided an indirect measure of the number of successful copulations. In a third analysis, reproductive success was evaluated as the total number of offspring sired by each individual male. For the latter two analyses, we used a mixedmodel analysis of variance to evaluate the effects of incubation temperature (independent variable) on reproductive success (dependent variable); field enclosure was included as a random effect. We used multiple regression analysis to evaluate relationships between our measures of behavior based on the four PC scores and reproductive success. Because we measured individual reproductive success, we could evaluate selection gradients for behavior. Standardized coefficients for linear (β) and quadratic (γ) relationships with reproductive success provide a measure of the strength and form of selection on behavior (Lande and Arnold 1983). To evaluate linear (i.e., directional) selection on behavior, all four PC scores were used as independent variables with reproductive success (i.e., standardized number of offspring sired) as the dependent variable in a multiple regression. To evaluate quadratic selection (i.e., stabilizing or disruptive) on behavior, these analyses were rerun including the square of each independent variable with number of offspring sired (standardized) as the dependent variable. All PC scores were standardized to a mean of zero and unit variance (i.e., z scores).

Incubation and morphological determinants of male behavior We detected no influences of incubation temperature on male responses to a standard opponent, regardless of the type of analysis employed. Incubation treatment had no effect on whether or not individuals responded to the stimulus during any trial (chi-square tests, trial 1: χ2 = 1.2, df=2, P=0.385; trial 2: χ2 =1.2, df=2, P=0.385; trial 3: χ2 = 2.7, df = 2, P=0.257). We found no treatment differences in the number of aggressive (χ2 =1.8, P= 0.411), submissive (χ2 =0.2, P=0.910), or exploratory (χ2 =3.6, P=0.167) behaviors in response to the video stimulus. Similarly, the analyses based on PC scores showed non-significant effects of incubation temperature on behavior (PC1: χ2 =3.2, P=0.203; PC2: χ2 =1.2, P= 0.539; PC3: χ2 =2.3, P=0.315; PC4: χ2 =1.7, P=0.438). Our analyses also revealed no effect of incubation temperature on any individual response type (all P values>0.05). Latency of responses to the stimulus (i.e., minutes until first response) also was unaffected by incubation temperature (χ2 =0.6, P=0.740). For males produced at the 27°C incubation temperature, the application of the aromatase inhibitor had no effect on behavior (PC1: χ2 =4.9, P=0.482; PC2: χ2 =0.2, P=0.688; PC3: χ2 =0.2, P=0.688; PC4: χ2 =0.7, P=0.421). The morphological variables that we measured were poor predictors of behavioral responses (Table 2). Neither male body size nor head dimensions were significantly related to behavior. Tail length relative to SVL was the only trait that significantly predicted behavior. When behaviors were represented as PC scores, individuals with relatively long tails showed fewer exploratory behaviors than those with short tails. However, when P values (in Table 2) were adjusted for multiple comparisons using Bonferroni corrections, no relationship between morphology and behavior was statistically significant (adjusted alpha=0.01).

Results Most individuals (83%) responded to the video stimulus during at least one trial. Behaviors were repeatable across the three trials for most display types; that is, individuals that displayed aggressively (i.e., high number of tail flicks, backward-forward arm waves, pushupbody-rocks, and gular extenstions) remained aggressive (r values>0.42; P values0.43, P values0.545).

Incubation and behavioral determinants of reproductive success As previously reported for a larger sample of lizards (Warner and Shine 2008a), incubation temperature affected male reproductive success; this pattern was also evident for the subsample used in the current study (Fig. 1). Males from the intermediate incubation temperature (27°C) sired more offspring (F2,34 =3.4, P=0.045), contributed to more clutches (F2,34 =3.5, P=0.041), and had more successful copulations (χ2 =11.9, P=0.003) than did males from the cool (23°C) or warm (33°C) incubation treatments. However, display behavior was not correlated with male reproductive success (Table 3), and therefore neither linear nor quadratic selection on behavior was significant.

Behav Ecol Sociobiol Table 2 Statistical relationships between morphology and behavior in male jacky dragons (Amphibolurus muricatus) Independent variable

Territorial behavior PC 1

PC 2

PC 3

PC 4

Hatch date Snout-vent length Tail length Jaw length Jaw width

β=−0.04, P=0.810 β=−0.07, P=0.688 β=0.13, P=0.461 β=0.19, P=0.279 β=0.11, P=0.616

β=0.01, P=0.931 β=−0.11, P=0.457 β=−0.37, P=0.023 β=−0.06, P=0.710 β=−0.15, P=0.437

β=0.06, P=0.724 β=−0.09, P=0.611 β=−0.07, P=0.668 β=−0.06, P=0.715 β=−0.18, P=0.390

β=0.10, P=0.554 β=−0.19, P=0.262 β=0.08, P=0.643 β=0.10, P=0.556 β=−0.18, P=0.377

Head depth

β=−0.15 P=0.494

β=−0.10, P=0.615

β=0.23, P=0.300

β=0.22, P=0.304

Analyses were carried out with separate multiple regressions for each behavior (represented as principal component (PC)) as a dependent variable. PC1 aggressive behaviors, PC2 exploratory behaviors, and PC3 and PC 4 submissive behaviors; β standardized estimates from the multiple regressions

Discussion The link between behavior and reproductive success is critical for understanding how selection has shaped the evolution of complex display behaviors. Sexual-selection theory assumes that variation in male displays in a population should influence variation in mating success, and hence fitness. In this study, we address this widespread assumption, but find no support for a link between male display behavior and reproductive success in the jacky dragon. Importantly, by incubating eggs under controlled conditions, raising lizards in semi-natural field enclosures, and using a standardized method for evaluating adult male behavior, we were able to remove numerous confounding factors that would have otherwise hindered our ability to detect links between adult male behavior and reproductive

(a) Males that sired offspring (%)

100 80 60 40 20 0 23

27

33

Number of offspring sired

(b) 12 Table 3 Selection gradients on behavioral phenotypes of male jacky dragons (Amphibolurus muricatus)

10

Independent variable

8

Linear (β)

Quadratic (γ)

PC 1 PC 2 PC 3

β=−0.06, P=0.717 β=0.12, P=0.483 β=0.03, P=0.827

γ=−0.36, P=0.328 γ=0.36, P=0.477 γ=−0.38, P=0.519

PC 4

β=0.19, P=0.238

γ=0.12, P=0.775

6 4 2 0 23

27

33

Incubation temperature (oC) Fig. 1 Effect of incubation temperature on reproductive success of male jacky dragons (Amphibolurus muricatus). a Reproductive success is classified as males that sired offspring versus those that did not. b Reproductive success is measured by the number of offspring sired by individual males. Error bars represent 1 SE. The same patterns were evident when reproductive success was evaluated as the number of clutches to which each male contributed

Selection gradient

Reproductive success was measured as the number of offspring sired by individual males. Two separate multiple regression analyses were carried out to quantify the intensities of linear (β) and quadratic (γ) selection on behavioral traits; γ=coefficient from polynomial regression multiplied by two Territorial behaviors are represented by principal components (PC; see Table 1). PC 1 aggressive behaviors, PC2 exploratory behaviors, and PC 3 and PC4 submissive behaviors. The same patterns were evident when reproductive success was indexed as the number of clutches to which each male contributed


success. For example, factors such as ambient temperature, rainfall, and presence of other lizards or prey would have been difficult, if not impossible, to control if our behavioral trials were conducted in the field or in the semi-natural enclosures in which lizards were raised. Selection on behavior Behaviors associated with territory defense are critical components of male fitness in polygynous species (Fox 1978, 1983; Fitze et al. 2008). Males that competitively exclude rivals from their territories tend to have greater access to females (Brown and Orians 1970), and thereby enhance their reproductive success. Thus, behaviors that improve an individual’s ability to defend territories are expected to be favored by sexual selection. In jacky dragons, the complex repertoire of display behaviors presumably is important in defending territories, which should influence male mating success as shown in other species (Andersson 1994; Lacey and Wieczorek 2001; Bergman et al. 2007). Surprisingly, however, we found no association between male display behaviors and reproductive success in our study. Individual males that tended to respond with submissive displays were just as successful at siring offspring as those that responded aggressively. Because jacky dragons have evolved a suite of complex displays, these non-significant results contradict our expectations. Indeed, numerous studies on a variety of animal species have concluded that more aggressive individuals have greater success at obtaining quality territories resulting in enhanced survival and reproductive success compared to submissive individuals (Tubbs and Ferguson 1976; Fox 1983; Reaney and Backwell 2007). We propose three potential explanations for the lack of association between male–male behavior and reproductive success found in this study. First, territory size in nature is likely to be larger than in the enclosures in which jacky dragons were housed, and therefore male and female densities were unnaturally high. These high densities could potentially enhance the success of submissive males. Under natural conditions, controlling a territory may be a more important predictor of reproductive success as suggested in other lizard species (Fox 1978; Ferguson et al. 1983; but see Lebas 2001). Second, our behavioral measurements focused only on responses to other male lizards. Although such responses are important for maintaining territories, female choice may be just as important in obtaining mates (Martín and López 2000, 2006; Stapley 2008; but see Olsson and Madsen 1995; Olsson 2001). Indeed, displays used in male–male interactions also may be used in courtship (Crews 1975) suggesting that interpretations of display behavior may vary depending on context. The submissive displays observed in our study (male–male

interactions) may have a different meaning during male– female interactions and, therefore, could obscure the expected relationship between display behavior and reproductive success. Third, males of many lizard species exhibit polymorphisms in reproductive strategies (Sinervo and Lively 1996; Sinervo et al. 2007). For example, submissive individuals may exhibit different but equally effective strategies that enable them to obtain as many copulations or fertilizations (e.g., due to differences in ejaculate size or sperm quality) as do aggressive individuals. The possibility of such polymorphism in male reproductive strategies in jacky dragons has not been investigated. Incubation effects on behavior and reproductive success Incubation temperature is known to influence a variety of offspring traits, including fitness-relevant behaviors (Qualls and Andrews 1999; Downes and Shine 1999; Freedberg et al. 2004). However, little is known about the long-lasting effects of incubation temperature, especially after offspring reach adulthood. In our study, the temperature that embryos experienced during development had no effect on adult male behaviors. Our results contrast with those found in another lizard species with TSD (leopard gecko; Eublepharis macularius), where female-producing temperatures have feminizing effects on adult behavior, and temperatures that produce predominantly males tend to have masculinizing effects (Flores et al. 1994; Flores and Crews 1995; Crews et al. 1998). Moreover, incubation temperature may affect brain organization during development in ways that influence aggressive behavior (Coomber et al. 1997). Given these sex-specific effects in E. macularius, males from eggs incubated at female-producing temperatures are expected to have lower reproductive success than the aggressive males produced at other incubation temperatures. Hence, selection should favor TSD because it would mean that males are produced at temperatures that result in aggressive behavior (i.e., a behavior that is more beneficial to male fitness than to female fitness). This result for E. macularius is consistent with theoretical models for the adaptive significance of TSD (Charnov and Bull 1977). Although jacky dragons have TSD, and incubation temperature has sex-specific effects on life-time reproductive success (Warner and Shine 2008a), we found no links between incubation temperature and male behavior in this study. The contrasting results of the current study with those of previous research on E. macularius suggest that the mechanisms involved in generating sex-specific temperature effects on fitness are complex, and may vary among species that exhibit TSD. How, then, does incubation temperature exert its effect on male reproductive success? Factors other than male territorial behavior may mediate the thermal effects on


reproductive success. For example, previous work shows that eggs incubated at warm incubation temperatures hatch early in the season, and individuals from these eggs (from 27 to 33°C incubation temperatures) are relatively large at the onset of the first and subsequent reproductive seasons (Warner and Shine 2005, 2008a). However, this effect of incubation temperature on body size does not explain why males from the 33°C incubation temperature have lower reproductive success than those produced at 27°C because males from these two incubation temperatures do not differ in body size (Warner and Shine 2005, 2008a). Other potential mechanisms may involve effects of incubation temperature on female mate choice, male attractiveness, or sperm quality or competition. Further research into the mechanism(s) through which incubation temperature affects male reproductive success is needed to better understand the factor(s) driving the evolution of TSD in this species.

Conclusions The current study, as well as our previous work on the same captive population of jacky dragons (Warner and Shine 2005, 2008a) demonstrated that incubation temperature affects male reproductive success. Our study enabled us to test the idea that this pattern is mediated through an incubationtemperature effect on male social behavior; but based on display behaviors measured during male–male interactions, we found no support for this hypothesis. Moreover, our results provide no evidence that display behaviors used during male–male interactions impact reproductive success in the jacky dragon. These findings fail to support the common assumption that display behaviors associated with territoriality impact male reproductive success. Instead, perhaps selection has shaped the complex display patterns of the jacky dragon under different conditions. For example, these display patterns may become important for male reproductive success during interactions with females (e.g., Murai and Backwell 2006), or under different environmental circumstances (Snekser et al. 2009). The role of male display behaviors in generating variation in fitness is complex and is likely to vary under different contexts. Acknowledgments We thank M. Elphick, P. Harlow, S. Ruggeri, T. Schwartz, F. Seebacher, J. Thomas, and M. Thompson for assistance. Thanks to members of the Janzen and Bronikowski labs for comments on an early draft of this manuscript. DAW was supported by an International Postgraduate Award and by an International Postgraduate Research Scholarship. KLW was supported by the Macquarie University CISAB Postgraduate Award and an Australian Research Council Discovery Grant to CSE. DVD was supported by a Research Award in Areas and Centres of Excellence. Further research funding was provided by Sigma Xi, the American Society of Ichthyologists and Herpetologists, the Norman Wettenhall Foundation, the Linnean Society of New South Wales, the Society for Integrative and

Comparative Biology, the Chicago Herpetological Society, the Royal Zoological Society of New South Wales (to DAW), and the Australian Research Council (to RS). Lizards were collected under permit #S10658 of the New South Wales National Parks Service. All protocols for this research were approved by The University of Sydney Animal Care and Ethics Committee (#L04/10-2003/3/3818) and by the Macquarie University Animal Care and Ethics Committee (#2003/025 and #2003/014).

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