J Comp Physiol A DOI 10.1007/s00359-009-0437-4

ORIGINAL PAPER

Sex steroid correlates of female-speciWc colouration, behaviour and reproductive state in Lake Eyre dragon , maculosus

Tim S. Jessop · Rita Chan · Devi Stuart-Fox

Received: 28 October 2008 / Revised: 16 March 2009 / Accepted: 18 March 2009 © Springer-Verlag 2009

Abstract In some , females develop bright colour- females to retain high steroid levels for inducing rejection ation to signal reproductive status and exhibit behavioural behaviours. repertoires to incite male courtship and/or reduce male harassment and forced copulation. Sex steroids, including Keywords Female reproduction · Sex steroids · progesterone and testosterone, potentially mediate female Female colouration · Mate rejection · reproductive colouration and reproductive behaviour. We measured associations among plasma proWles of testoster- Abbreviations one and progesterone with variation in colour expression JND Just noticeable diVerence and reproductive behaviour, including unique courtship SE Standard error rejection behaviours, in female Lake Eyre dragon lizards, SD Standard deviation (Ctenophorus maculosus). At onset of breeding, progester- LWS Long wavelength sensitive one and testosterone increased with vitellogenesis, coinci- GLMM General linear mixed model dent with colour intensiWcation and sexual receptivity, LSD Least squared diVerence indicated by acceptance of copulations. As steroid levels peaked around the inferred ovulation time, maximal colour development occurred and sexual receptivity declined. Introduction When females were gravid and exhibited maximal mate rejection behaviours, progesterone levels remained consis- Conspicuous colours and ornaments are much less common tently high, while testosterone exhibited a discrete second in females than males and consequently, studies of the adap- peak. At oviposition, signiWcant declines in plasma steroid tive function and proximate basis of female ornamentation levels, fading of colouration and a dramatic decrease in are comparatively rare (reviewed in Amundsen 2000; male rejection behaviours co-occurred. Our results indicate Amundsen and Parn 2006). Although female ornamentation a generally concordant association among steroid levels, is typically a reduced version of that in males, many species colouration, behaviour and reproductive events. However, possess colour patterns and ornaments that are speciWc to the prolonged elevation in progesterone and a second peak females (e.g. Amundsen and Forsgren 2001; Baird 2004; of testosterone was unrelated to reproductive state or fur- Cooper and Greenberg 1992; Cuadrado 2000; Heinsohn et al. ther colour change, possibly suggesting selection on 2005; Kraaijeveld et al. 2004; Montgomerie and Thornhill 1989; Nunn 1999; Roulin et al. 2001; Rowland et al. 1991; Watkins 1997; Weiss 2006). Female-speciWc ornamentation T. S. Jessop Department of Wildlife Conservation and Science, may have several adaptive functions including signalling Zoos Victoria, Parkville 3052, Australia reproductive maturity, status and genetic quality to both mates and potential female competitors (Amundsen and Parn & R. Chan · D. Stuart-Fox ( ) 2006; Cooper and Greenberg 1992; Nunn 1999). With Department of Zoology, University of Melbourne, Melbourne 3010, Australia respect to signalling reproductive status, females may signal e-mail: [email protected] the most eVective time for fertilisation to stimulate male 123 J Comp Physiol A courtship or incite male–male competition (reviewed in Coo- by seasonal or context dependent increases in androgens per and Greenberg 1992; Nunn 1999) and/or signal non- may be evident (Desjardins et al. 2006; Gill et al. 2007; receptivity in order to decrease the direct costs of male Hegner and WingWeld 1986; Langmore et al. 2002; harassment and mating (Cooper and Greenberg 1992). Woodley and Moore 1999a). Complementing variation in colour expression are often Lizards are good model organisms for understanding the distinctive shifts in behavioural repertoires that reXect a physiological and particularly the endocrine basis of both female’s transition between receptive and non-receptive female-speciWc colouration and associated behavioural breeding states (Cooper and Greenberg 1992; Nunn 1999). transitions because in several species, the development, Changes in receptivity are typically associated with diVer- pattern and intensity of female colouration and correspond- ences in a female’s response towards male courtship and ing behavioural repertoires may be directly regulated by copulation attempts. When receptive, females may readily sex steroids (see also Calisi and Hews 2007; reviewed in court and mate with males, whereas non-receptive females Cooper and Greenberg 1992). Previous studies have dem- often actively avoid male interest by using defensive and, onstrated that progesterone and testosterone, and to a lesser in some instances, highly aggressive behaviour constituting degree oestradiol cause development of orange pigments in courtship rejection (Berger 1983 [horses]; Cooper and adult female lizards (Cooper and Crews 1988; Cooper and Greenberg 1992 [lizards]; Farr 1980 [Wsh]; 1984 [birds]; Ferguson 1972a, b; Medica et al. 1973). Similarly, adminis- McKinney et al. 1983; Nunn 1999 [primates]). For the lat- tration of progesterone and testosterone to ovarectomised ter, courtship rejection behaviours may function to prevent propinqua reinstated aggressive courtship forced male copulation and reXect potentially costly female rejection behaviours (Cooper and Crews 1987). Other stud- resistance strategies that have evolved under sexual conXict ies on female lizards show correlative evidence that steroid (reviewed in Arnqvist and Rowe 2005; Chapman et al. levels inXuence both colouration and aggressive behaviours 2003; Clutton-Brock and Parker 1995). (Calisi and Hews 2007; Chan and Callard 1974; Cooper Although the evolution of female resistance strategies et al. 1983; Cooper and Clarke 1982; Cooper and Crews has received recent attention (reviewed in Arnqvist and 1988; Salvador et al. 1997; Watt et al. 2003; Weiss et al. Rowe 2005; Chapman et al. 2003; Clutton- Brock and 2002; Woodley and Moore 1999a, b). Parker 1995), our understanding of the proximate physio- We investigated steroid levels in relation to female col- logical mechanisms (e.g. steroid hormones) potentially our expression and reproductive behaviour in the Lake Eyre mediating courtship rejection behaviours in females is dragon lizard, in which females exhibit both distinctive still poorly deWned (Cooper and Crews 1988). This is in reproductive colouration and unique courtship rejection contrast to males where there is strong theoretical frame- behaviours. The species is restricted to a few large salt pans work that links the frequency and intensity of aggression in Australia’s arid southern interior and shelters in loose within a reproductive context and its mediation by sex sand beneath the salt crust (Mitchell 1973; Olsson 1995b). steroid hormones, particularly testosterone (WingWeld Females develop extensive, conspicuous, ventro-lateral et al. 1990). SpeciWcally, in male vertebrates seasonal orange colouration (Fig. 1b) during breeding while males plasma proWles of testosterone may be distinctly diVerent remain cryptically coloured (Mitchell 1973). Males com- between monogamous and promiscuous males (Wing- pete vigorously for access to territories and females and Weld et al. 1990). This diVerence forms the basis of the exhibit persistent courtship, harassment and attempted challenge hypothesis (sensu WingWeld et al. 1990), forced copulations (Olsson 1995a). Female colouration which predicts that in promiscuous males exhibiting little stimulates male courtship and is also emphasised during paternal care, plasma levels of testosterone remain ele- rejection displays yet there is no marked diVerence in vated to facilitate ongoing aggression, whereas mono- colouration between receptive and unreceptive females gamous males have initially high testosterone levels (Chan et al. submitted). Coinciding with this colouration, followed by a noticeable decrease coinciding with the female Lake Eyre dragons have evolved a unique sequence onset of paternal behaviours (WingWeld et al. 2001). of rejection behaviours. Initially, females either Xee or per- However in monogamous males, androgen levels may form lateral displays, elevating themselves on all four legs, increase dramatically during certain aggressive contexts while laterally compressing the body to reveal their ventro- (e.g. territorial intruders, nest predators) but decrease lateral orange colouration. As a last resort, however, quickly as the challenge passes and as the need to tend females Xip themselves onto their backs exposing the bright oVspring remains. Several studies support an analogous orange ventral colouration (a behaviour unknown in any behavioural modiWcation of seasonal androgen proWles in other lizard) because this position prevents forced male females and again suggest that depending on amount of intromission (Olsson 1995a). aggression exhibited during the female reproductive As an initial step towards understanding the proximal period, distinct proWles in plasma testosterone characterised regulation of the conspicuous colouration and remarkable 123 J Comp Physiol A

Fig. 1 a A non-reproductive fe- male exhibiting white colour- ation and b a female exhibiting orange colouration. ReXectance measurements were taken from three body regions: the orange throat (1, 2), black gular stripe (3) and orange abdomen (4, 5, 6, 7)

reproductive behaviours of female Lake Eyre dragons, we Methods examined the correlation between plasma levels of proges- terone and testosterone and female colour expression and capture and husbandry reproductive behaviour. First, we assessed if and how these hormones were correlated with development and subse- We captured 57 lizards (25 females, 32 males) by hand quent Wne-scale temporal variation in colour expression from Lake Eyre, South Australia (28.95–29.05°S, 137.65– both across the breeding period and within individual 137.76°E). The were captured shortly after females. We determined patterns of colour signal expres- emergence from hibernation (between 22 August and 7 sion as perceived by the lizard, rather than human visual September 2007) and none of the females showed orange system by quantifying spectral reXectance of colour pat- colouration until several weeks after being brought into terns and applying a model of colour perception based on captivity. We measured body size (snout-vent length, SVL) visual pigment sensitivities for a congeneric species to the nearest mm at capture and 1 month after being (Barbour et al. 2002). Next, we assessed the degree of con- brought into captivity and used the mean of these in subse- cordance between steroid proWles and transitions in female quent analyses. The mean (§ SD) size of females in this reproductive behaviour. SpeciWcally, we tested whether study was 56.6 § 4 mm (range 49–64 mm) and males were plasma steroid proWles diVered in females as they pro- 65.2 § 3.5 mm (range 56–70 mm), which is larger than the gressed from receptive to non-receptive states, when minimum size at sexual maturity (45 mm for females and females show a marked increase in mate rejection beha- 55 mm for males; Mitchell 1973). viours. If in Lake Eyre dragons steroid hormones regulate We transferred lizards to the animal facility at the three interacting components of female reproduction (e.g. University of Melbourne where they were housed indi- physiology, colouration, behaviour), we might expect to vidually in 61 £ 30 £ 30 cm glass tanks containing a measure distinctive proWles in one or both plasma steroids layer of sand and salt crust to mimic natural habitat, and (i.e. relative to female lizards where colouration or male separated by opaque partitions. The room was main- rejection behaviours are absent). Given persistent male tained at an average temperature of 28°C on a 12:12 harassment and forced copulation, female Lake Eyre drag- day:night cycle, and a heat lamp was suspended above ons are predicted to be a likely candidate to exhibit elevated each tank to allow animals to attain their preferred body plasma steroid levels to facilitate ongoing maintenance of temperatures. Lizards were misted and fed live crickets male rejection behaviours until oviposition. (Anchieta domesticus) or mealworms (Tenbrio spp. 123 J Comp Physiol A larvae) dusted in calcium and multivitamins three times Table 2 Sample sizes for measurements of female colouration a week. (spectrometry and area) and male, female behaviour and plasma steroid concentrations Sampling design Female Spectrometry Reproductive Plasma steroid stage (N) behaviour (N) concentrations (N)

Our aim was to examine changes in plasma concentrations 0 14 (13) 32 (16) 20 (17) of progesterone and testosterone in relation to female col- 1 7 (7) 18 (9) 9 (8) ouration and behaviour throughout the reproductive cycle, 2 10 (8) 19 (10) 6 (5) which we divided into nine stages (Table 1). To minimise 3 12 (10) 19 (10) 13 (11) stress to the animals, we did not measure colouration and 4 19 (14) 20 (10) 7 (6) behaviour or take blood samples of each individual within 5 7 (7) 20 (10) 5 (5) every stage. Instead, we aimed to obtain between Wve and 6 8 (7) 20 (10) 7 (7) ten sets of colour measurements and blood samples and 7 10 (9) 18 (9) 8 (5) approximately 20 behavioural trials for females represent- ing each stage (sample sizes in Table 2). Within an ovarian 8 11 (11) 20 (10) 7 (7) cycle, we took colour measurements and blood samples of Total 98 186 82 females at an average of 2.65 (§ 1.2 SD, range 1–5) out of The same individual was used in multiple stages. In each case, the the nine stages and used females in no more than two number of measurements is given with the number of females repre- sented in brackets behavioural trials per stage. We took a total of 98 sets of reXectance measurements, conducted 186 behavioural trials and took 82 blood samples (Table 2). For a subset of reproductive cycle elsewhere (Chan et al. submitted), there- females, we had both blood samples and colour measure- fore here we provide only a brief description of methods for ments (N = 57) for the same stage, allowing us to correlate obtaining colour and behavioural data, as well as how col- plasma steroid concentrations with female colour intensity our and behaviour vary across the ovarian cycle below. at the individual level. We did not take blood samples immediately before or after behavioural trials. As our aim Female reproductive stages in this study was to examine changes in steroid levels in relation to colouration and behavioural changes across the To quantify plasma steroid concentrations in relation to reproductive cycle, we took blood samples within the same female colouration and behaviour we divided the reproduc- reproductive stage, that is, within 3 days of behavioural tri- tive cycle into nine stages (Table 1). These stages were als. The temporal variability in blood sampling relative to deWned in a way that would enable us to detect Wne scale behavioural trials precluded us from testing for correlations temporal changes in colour intensity, behaviour and plasma between progesterone or testosterone and behaviour at the steroid concentrations across the reproductive cycle, since individual level. We have described how female colour precise reproductive status (e.g. timing of ovulation) cannot expression and reproductive behaviour vary across the be determined non-invasively in this species. Stage 0 was

Table 1 Stages deWning female Stage DeWnition Colouration Reproductive status colour and associated reproduc- tive state in Lake Eyre dragons 0Prior to Wrst developing orange White Non-reproductive 1 0–4 days after orange developed Orange Vitellogenic follicles not always detectable via palpation 2 5–8 days after orange developed Orange Vitellogenic follicles 3 9–12 days after orange developed Orange Ovulation most likely between stages 3 and 4 4 13–16 days after orange developed Orange Shelling oviductal eggs by the end of stage 4 at the latest 5 17–20 days after orange developed Orange Shelling oviductal eggs 6 21 days after orange developed to laying Orange Shelled oviductal eggs after orange developed 7 0–10 days post-laying Faded Post-oviposition orange/white 8 10+ days post-laying without Faded Post-reproductive orange re-intensifying orange/white

123 J Comp Physiol A the period at the beginning of the breeding season, where detailed in Siddiqi et al. (2004). We used data on the spec- female colouration was white (Fig. 1a). Some females tral sensitivities of a congeneric lizard, Ctenophorus orna- remained white (N = 7) and never developed orange colour- tus (Barbour et al. 2002). We assumed that as in other ation or laid eggs (fertilised or unfertilised) over the entire vertebrates, the three single cones are used for chromatic breeding season (Table 1), while the remaining 18 females (colour) discrimination, while the long wavelength sensi- developed orange colouration and laid eggs. Stages 1–5 tive (LWS) photoreceptors in the double cones are used for consisted of 4-day increments, from the date females deve- achromatic (brightness) discrimination (Osorio and Voro- loped orange colouration, and stage 6 extended to laying byev 2005). date (Table 1). Vitellogenic follicles could be detected via To assess female behaviour over the reproductive cycle, palpation during stages 2 and 3, while females had shelled we conducted behavioural trials during the lizards’ breed- oviductal eggs from stages 4 or 5 until laying. During these ing season from September 2007 to January 2008. We six stages females retained their orange colouration placed females in male tanks, videotaped the interaction for (Fig. 1b; Table 1). Stage 7 was deWned as the 10 days fol- approximately 30 min and scored female reproductive lowing laying and stage 8 was the period greater than behaviours from the video footage. Behavioural interac- 10 days after laying without the female becoming reproduc- tions generally began with the male courting the female tively active again, with colouration in these two stages with a series of head bobs and push-ups. In response, being either a faded orange or white (Table 1). females would perform appeasement behaviours, consisting We also deWned three broader reproductive categories: of head bobs and arm waves, allow copulation, Xee or reject (1) “non-reproductive” comprising females that were yet to the male by Xipping over or using a lateral display, some- develop orange colouration (stage 0), post-oviposition times performed along with a slow stiVened walk. Arm (stage 7) and post-reproductive (stage 8); (2) “receptive” waves consisted of a single extension of the forearm in a comprising females from the period of developing orange lateral forward circular motion; lateral displays consisted of colouration (stage 1) until acceptance of their last females elevating their bodies by extending all four legs (unforced) copulation (derived from behavioural trials— beneath them, lateral compression of body and arching of see below) and (3) “unreceptive” comprising females from the back while Xips consisted of the female Xipping herself the date of their last (unforced) copulation until oviposition. onto her back exposing her bright orange ventro-lateral col- These categories correspond to major changes in female ouration (Fig. 1). We scored the number of head bobs, reproductive behaviour. female arm waves, copulation duration, and the duration of lateral displays and Xips. We converted all behaviours to Female colouration and behaviour measures per minute of active trial time, deWned as the time from when the individuals Wrst interacted and excluding the We provide detailed methods describing measurement of time that each lizard spent hiding beneath the salt crust. female colouration and behaviour elsewhere (Chan et al. Behavioural data were log-transformed to meet model submitted). BrieXy, we quantiWed the spectral properties of assumptions. female colouration at each reproductive stage by taking reXectance readings (USB 4000 spectrometer and PX-2 Blood sampling protocols light source Ocean Optics, Dunedin, USA) in the range of 300–700 nm, the visual spectrum for most diurnal lizards To standardise possible circadian variation in steroid hor- (Loew et al. 2002). We measured three body regions: the mone levels, blood was collected between 1200 and throat, black gular stripe down the centre of the throat and 1400 hours from 31 adults (25 females, 6 males; Table 2) abdomen (Fig. 1). As visual systems encode signals as con- using 100 l heparinised capillary tubes sampling from the trasts between adjacent colours, we analysed colour varia- sinus angularis, which was accessed using a needle tip tion as changes in the contrast between orange (throat and through the corner of the mouth, following the standard abdomen) or black (gular stripe) colouration against the procedure for ‘small’ lizards (Olsson et al. 2000; Woodley white ventral surface, as perceived by the lizard visual sys- and Moore 1999a). Blood samples were kept on ice, centri- tem. We applied the model of Vorobyev and Osorio (1998), fuged within the hour at 10,000 rpm for 2 min and the which has been applied to a range of vertebrates (Hemmi plasma was separated and stored in a freezer at ¡20°C until et al. 2006; e.g. Siddiqi et al. 2004; Stuart-Fox and Mouss- assayed. Individual blood sampling occurred no more than alli 2008 and references therein). It assumes that visual dis- once a week, was dependant on individual health status,  crimination is limited by photoreceptor noise, i, and can and collected with a focus on obtaining samples from 5 to be used to estimate the discriminability of two colours in 10 representatives for each stage, rather than a sample per units of discrimination thresholds or just noticeable diVer- female per stage (Table 2—see Sampling design). These ences (JNDs). We used the same model calculations as blood sampling procedures have been shown to not 123 J Comp Physiol A permanently aVect small lizards, with individuals perform- account for correlated data due to repeated use of individu- ing normal behaviours (e.g. eating) within minutes after als and use of the same individual over more than one collection (Chan, pers. obs.; Whiting et al. 2006). reproductive cycle, we included female ID and female cycle (Wrst, second or third) nested within female ID as ran- Plasma steroid analysis dom factors in GLMMs. We used least square diVerences (LSD) post hoc tests to test for diVerences between each We measured total plasma concentrations of progesterone pair of stages. We also tested whether progesterone or and testosterone using commercially available ELISA kits testosterone concentrations diVered between females in the (Caymen Chemical, Michigan, USA) and their associated three broader reproductive categories (non-reproductive, protocols. Samples were analysed across three assays per receptive and unreceptive). hormone, with assays also containing a small number of If steroid levels are a potential physiological basis for male samples to serve as a negative and positive control for explaining stage and individual based diVerences we progesterone and testosterone, respectively. Preliminary might expect to observe signiWcant correlations between assays determined that the suYcient volume of sample steroid level and our measures of colouration and behav- plasma to be used for assay was 10 l for progesterone and iour. Thus, we calculated mean values of steroid levels, 20 l for testosterone (a minimum of 5 l used in samples colour and behavioural traits and performed multiple where minimal plasma was available and volume correc- regressions with each colour or behavioural trait as the tions were performed in calculations). To reduce cross reac- dependent variable and both progesterone and testosterone tion and interference all samples were individually as predictors. To assess which steroid best explained extracted under the steroid speciWc solvent ratios of Ethyl variation in colouration or behaviour, we applied step- Actetate:Hexane (5:95) for progesterone (following Ren- wise model selection with a criterion for a variable free et al. 1994) and Hexane:Toluene (2:1) for testosterone remaining within the model set at P < 0.1, and, if both (following Williamson et al. 1990). To measure the variables were retained, compared partial R2 values. eYciency of extraction 20 l (t 2,000 cpm) of tritiated ste- Finally, for the subset of females for which we had both roid was added to each sample prior to extraction. To esti- blood samples and colour measurements within the same mate steroid extraction eYciency, 50 l of each extracted reproductive stage, we tested for a correlation at the sample was placed into a scintillation vial containing 2 ml individual level between plasma concentrations of pro- of scintillation Xuid (Ultima Gold). Sample radioactivity gesterone or testosterone and female colouration, again was estimated using a Beckman 2100R Liquid Scintillation using GLMMs. Counter. The extraction eYciency for each sample was cal- culated as the quotient of radioactivity (CPM) remaining in the sample relative to the total amount of radioactivity Results added to each sample pre-extraction (determined from extraction controls). Variation in colouration and behaviour Final steroid concentrations (measured in duplicate) were calculated from standard curves and corrected for Female colour expression changes signiWcantly across the individual sample recovery, individual plasma volume and reproductive cycle, reaching its full intensity from stages 2 the addition of tritiated steroid. For progesterone assays, to 5 before fading just prior to laying or in stage 7 just after average (§SE) female recovery was 65.7% § 0.028 with laying (Fig. 2; Chan et al. submitted). SpeciWcally, both the an intra-assay coeYcient of variation of 22.6% and an inter- achromatic and chromatic contrast of the throat and abdo- assay coeYcient of variation of 13.04%. For testosterone men against the adjacent white ventral surface increase rap- assays, average female recovery was 67.2% § 0.018 with idly between stages 1 and 2, reaching a plateau until stage 5 an intra-assay coeYcient of variation of 13.6% and an inter- before decreasing at stages 6 or 7. assay coeYcient of variation of 14.0%. The assay detection Female behaviours vary signiWcantly through the repro- limits were 0.15 and 0.10 ng/ml for plasma testosterone and ductive cycle (Chan et al. submitted; Fig. 3). Females per- progesterone, respectively. form arm waves and head bobs, which appear to function as appeasement behaviours, primarily when white and non- Statistical analysis reproductive (stage 0). Females begin accepting copulation shortly after showing orange colouration (stage 1). Copula- We tested whether female plasma concentrations of proges- tion frequency peaks during stage 2 (Fig. 3a) then falls terone and testosterone diVered signiWcantly between sharply through stage 3. From stage 4 to 6, females increase reproductive stages using a Generalised Linear Mixed the frequency of rejection behaviours—lateral displays Model (GLMM; PROC GENMOD SAS version 9.1). To (Fig. 3b) and Xipping over (Fig. 3c). 123 J Comp Physiol A

80 a) Gular stripe 1.0 a)

60 0.8

0.6 40 0.4 20 0.2

Achromatic contrast (JNDs) 0 Copulation duration (sec/min). 0.0

16 b) Throat 4 Abdomen b) 12 3

8 2 4 (sec/min)

1

Achromatic contrast (JNDs) 0 Lateral display duration

Throat 0 10 c) Abdomen Gular stripe 8 8 c) 7 6 6

4 5 4 2 3

Chromatic contrast (JNDs) 0 2 012345678 Flip duration (sec/min) 1 Female stages 0 Fig. 2 Mean (§SE) of a achromatic (brightness) contrast of the gular 012345678 stripe against the surrounding white throat and b achromatic contrast Female stage of the orange throat and abdomen against adjacent white body regions; Fig. 3 Mean (§SE) duration (in seconds per minute of trial time) of a c chromatic (colour) contrast of the throat, abdomen and gular stripe copulations and the rejection behaviours, b lateral display and c Xip against adjacent white body regions in units of discrimination thresh- over performed over the eight stages of the female reproductive cycle olds or just noticeable diVerences (JNDs)

levels at stages 0, 7 and 8 (LSD post hoc tests: P < 0.003 for each pairwise comparison). Variation in steroid levels In terms of broader reproductive categories, there were signiWcant diVerences among categories in concentrations

Levels of plasma progesterone and testosterone varied sig- of both steroids (progesterone F2,27 =18.28, P < 0.0001; W ni cantly throughout the reproductive cycle (progesterone testosterone F2,26 = 7.37, P =0.003; Fig.5). Non-reproduc- F8,44 = 8.11, P < 0.0001, testosterone F8,43 =4.65, tive females had signiWcantly lower levels of circulating P = 0.0004). Progesterone rapidly increased and remained plasma progesterone and testosterone than either receptive high in stages 3, 4 and 5 before dropping sharply prior to (progesterone F27 = 3.91, P = 0.0006; testosterone laying (Fig. 4). Mean values of progesterone in stages 3, 4 F26 = 3.59, P = 0.001) or unreceptive females (progester- W and 5 were signi cantly higher than in stages 0, 1, 7 and 8 one F2,27 =5.79, P < 0.0001; testosterone F26 = 2.75, (LSD post hoc tests: P < 0.007 for all pairwise compari- P = 0.011). There was a trend toward higher progesterone sons). Testosterone increased steadily until stages 2–3, after levels in unreceptive than receptive females (F2,27 = 1.86, which it decreased before another peak increase between P = 0.07) but no diVerence in testosterone levels between stages 5 and 6, followed by a rapid decline back to initial the two categories (F2,26 = ¡0.76, P = 0.45). levels at stages 7 and 8 (Fig. 4). The two testosterone peaks Multiple regressions of mean values at each stage at stages 2–3 and 6 were signiWcantly higher than Testosterone showed signiWcant relationships between both colour and 123 J Comp Physiol A

80 a) Neither steroid was associated with copulation frequency among stages.

60 At the individual level, there were clear correlations between plasma steroid concentrations and measures of colour intensity. Both measures of throat colouration were 40 signiWcantly correlated with both plasma progesterone con-

Progesterone centrations (achromatic contrast: F1,27 = 28.11, P < 0.0001; 20 chromatic contrast: F =20.81, P < 0.0001) and testo- concentration (ng/ml) 1,27 sterone concentrations (achromatic contrast: F1,27 = 10.68, 0 P < 0.003; chromatic contrast: F1,27 = 13.62, P = 0.001; 012345678 Fig. 6). There was also a strong relationship between the 120 achromatic contrast of the gular stripe and progesterone b) concentrations (F = 25.3, P < 0.0001) and a near-signiW- 100 1,27 cant trend with testosterone concentrations (F1,327 = 3.97, 80 P = 0.056). Similarly, for the abdomen, there was a consis- tent relationship between colour intensity and progesterone 60 concentrations (achromatic contrast: F1,28 = 27.25, 40 Testosterone P < 0.0001; chromatic contrast: F1,28 = 28.32, P < 0.0001) but not testosterone concentrations (achromatic contrast: concentration (ng/ml) 20 F1,28 =1.98, P = 0.17; chromatic contrast: F1,28 = 2.41, 0 P =0.13). 012345678 Female stage Fig. 4 Mean (§ SE) concentration of circulating plasma: a progester- Discussion one and b testosterone for each female stage across the reproductive cycle During breeding female Lake Eyre dragons exhibited dis- W 70 tinct patterns in plasma steroid pro les of both progester- Non-reproductive one and testosterone. At the onset of the breeding cycle, Receptive 60 plasma steroid levels rapidly increased and were directly Non-receptive 50 associated with the intensiWcation of colouration, cessation of appeasement behaviours and acceptance of male court- 40 ship. Steroid levels remained elevated throughout the 30 remainder of the cycle, including the transition from recep- tive to unreceptive status, until oviposition then rapidly 20 decreased to basal levels. During this period, colouration 10 also remained at maximum intensity. Decreasing progester- Hormone concentration (ng/ml) 0 one and testosterone levels around the time of oviposition Progesterone Testosterone coincided with diminished colouration and decreased male rejection behaviours. Hence our study reports a highly con- Fig. 5 Mean (§SE) concentration of circulating plasma progesterone W and testosterone in non-reproductive, receptive and unreceptive fe- cordant correlation between sex steroid pro les, female col- males ouration, mediation of speciWc behaviours and transition in reproductive state of female Lake Eyre dragons. behaviour and steroid levels (Table 3). Plasma progester- The close coincidence between initial increases in one level was a consistent predictor of colour expression, plasma steroid levels and colour development as well as showing a positive relationship with both the achromatic peak plasma progesterone levels and peak colour expres- and chromatic contrast of the throat and abdomen sion in female Lake Eyre dragons is consistent with pat- (Table 3). Testosterone level was a strong predictor of the terns found in other lizards. In other lizards, the frequency of arm waves and head bobs: in stages where development of bright female colouration also appears to these appeasement behaviours were frequent, testosterone be both directly regulated or correlated with elevated pro- values were concomitantly low (Table 3). The strongest gesterone levels. Plasma progesterone concentration was predictor of rejection behaviour (lateral display and Xip shown to be elevated during vitellogenesis in striped pla- duration) was progesterone, with a positive relationship teau lizards, Sceloporus virgatus, with a surge related to between progesterone levels and both behaviours (Table 3). ovulation (Weiss 2002). Brightly coloured female keeled 123 J Comp Physiol A

Table 3 Multiple regressions Dependent Variable Predictors retained Slope R2 P of mean values of each colour or in Wnal model behavioural trait at each stage with plasma progesterone and Throat achromatic contrast Progesterone + 0.61 0.013 testosterone levels as predictor variables Throat chromatic contrast Progesterone + 0.73 0.004 Gular stripe achromatic contrast None retained Abdomen achromatic contrast Progesterone + 0.43 0.056 Abdomen chromatic contrast Progesterone + 0.53 0.057 Arm wave frequency Testosterone ¡ 0.76 0.002 Head bob frequency Testosterone ¡ 0.86 0.003 Lateral display duration Progesterone + 0.68 0.006 Flip duration Progesterone + 0.60 0.01 Only variables retained (P <0.1) Number of copulations per trial None retained in the Wnal model are shown

250 Lacerta vivipara a) Testosterone common lizard, (Dauphinvillemant and Progesterone Xavier 1985). Our results strongly suggest that circulating 200 progesterone levels are related to colouration in Lake Eyre dragons, as orange coloured females possessed higher con- 150 centrations than white females and there was a highly sig- niWcant correlation between progesterone levels and colour 100 intensity at the individual level as well as based on mean levels among females across reproductive stages. 50 Across the female Lake Eyre dragon reproductive cycle,

Hormone concentration (ng/ml) testosterone remained elevated and exhibited two peaks, 0 1.4 6.4 11.4 16.4 21.4 one near the beginning of the reproductive cycle and the Achromatic contrast (JNDs) other near the end. The initial peak at the beginning of the reproductive cycle could facilitate multiple reproductive 250 Testosterone b) processes (e.g. oviductal hypertrophy) and associated col- Progesterone 200 our development (Chan and Callard 1974). Female spiny lizards, Sceloporus pyrocephalus, that exhibited colour- 150 ation possessed higher levels of plasma testosterone (Calisi and Hews 2007). Similarly, in both captive and wild 100 female-keeled earless lizards, Holbrookia propinqua, col- our expression is associated with elevated circulating tes- 50 tosterone (Cooper and Crews 1988). Additional evidence

Hormone concentration (ng/ml) for the role of testosterone in female colour expression 0 2.78 4.78 6.78 8.78 10.78 12.78 14.78 comes from experimental studies of exogenously implanted Chromatic contrast (JNDs) testosterone. Colouration was promoted by exogenously implanted testosterone in female eastern fence lizards, Fig. 6 Individual correlations between plasma concentrations of pro- gesterone and testosterone and intensity of throat colouration: a achro- Sceloporus undulatus, where females normally exhibit little matic (brightness) contrasts and b chromatic contrasts against adjacent or greatly reduced colouration compared to males (Cox white body regions in units of discrimination thresholds or just notice- et al. 2005; Quinn and Hews 2003). Testosterone has also V able di erences (JNDs). Thin black line is the trendline for progester- been implicated in female reproduction as female striped one and the thick black line is the trendline for testosterone plateau lizards, Sceloporus virgatus, appeared to have a surge of testosterone during ovulation (Weiss 2002). earless lizards, Holbrookia propinqua, also had higher lev- Although exact occurrence of ovulation could not be deter- els of progesterone than non-coloured females, both in the mined, the initial peak of testosterone in female Lake Eyre Weld and the laboratory (Cooper and Crews 1988). Proges- dragons may also have been associated with ovulation as terone levels inXuencing colouration can be produced from well as colour expression. The relationship between testo- both ovarian and non-ovarian sources (Cooper and Crews sterone and colour expression in Lake Eyre dragons was 1988). For example, the adrenal gland also produces pro- supported by higher testosterone levels in females that were gesterone during the reproductive cycle, as found in the reproductively active and exhibiting orange colouration 123 J Comp Physiol A than non-reproductive females and the signiWcant correla- Gill et al. 2007; Hegner and WingWeld 1986; Langmore tion between testosterone levels and throat colour intensity et al. 2002; Woodley and Moore 1999a). However the rela- at the individual level. tionships between sex steroids and female aggression are The second peak in testosterone (at stage 6) and the pro- diverse and potentially reXect the multiple contexts in longed elevation in progesterone is distinctive and we sug- which selection for female aggressive behaviours (includ- gest that both steroids could mediate and maintain ing mate rejection behaviours) can evolve. Consequently, a behaviours used to reject males (i.e. lateral displays and prolonged elevation in plasma progesterone and testoster- Xips) up until almost the end of the female’s reproductive one in Lake Eyre dragons may suggest a potential proximal cycle. Several lines of evidence support this assertion. First, response to mediate male rejection behaviours consistent the physiological and colouration aspects of reproduction with a prolonged period of ongoing male harassment post- are eVectively decreasing during this late stage and intui- receptivity. SuperWcially, Lake Eyre dragons exhibit sea- tively this should not require elevated testosterone and pro- sonal plasma sex steroid proWles consistent with those of gesterone levels to facilitate down regulation. Second, other male vertebrates exhibiting promiscuous mating systems, females lizards, including agamids, in which colouration high levels of aggression and limited parental care (Wing- and aggression are absent, exhibit decreases in these sex Weld et al. 1990). In other female vertebrates where aggres- steroids shortly after ovulation (Amey and Whittier 2000; sion is required but sporadic and typically coupled with Whittier and Tokarz 1992). Third, in other female lizards, periods of parental care, plasma testosterone is not chroni- testosterone and progesterone are directly implicated in cally elevated, but may increase rapidly during speciWc mediating female aggression, such as in the spiny lizard, aggressive contexts via behavioural androgen responses Sceloporus jarrovi (Woodley and Moore 1999a) and (Desjardins et al. 2006; Gill et al. 2007). These diVerences female-keeled earless lizards Holbrookia propinqua (Coo- in the interactions between behaviour and testosterone (and per and Crews 1987). Similarly, female marine iguanas, presumably other sex steroids) in relation to the relative fre- Amblyrhynchus cristatus, also exhibit elevated testosterone quency of aggression supports the applicability of the chal- levels following copulation coinciding with periods of post- lenge hypothesis to explaining the role of testosterone in mating aggression with males (Rubenstein and Wikelski female vertebrates (Desjardins et al. 2006; Gill et al. 2007). 2005). The speciWc role for testosterone in female aggres- However, recent studies have also suggested that testoster- sion is suspected to be indirect and via its aromatisation one levels of females can reXect a correlated response to into oestrogen at various sites including the ovaries, spine direct selection on testosterone levels in males, as opposed and/or brain (Woodley and Moore 1999b). For instance, to direct selection on females themselves (Ketterson et al. female lizards with experimentally increased or decreased 2005; Mank 2007). This is especially pertinent in the case oestrogen induced or reduced female aggression, respec- of female lizards where testosterone can be consistently tively (Woodley and Moore 1999b). Further there is some elevated across the breeding cycle. To demonstrate a non- evidence that progesterone may also inXuence female correlated response and provide evidence that elevated tes- aggression in leopard geckos, Eublepharis macularius tosterone in female Lake Eyre dragons is directly selected (Rhen et al. 1999), suggesting multiple hormones could for mediating female mate rejection behaviours we would contribute to mediating female aggression in lizards. In need to show an uncoupling between male and female sea- Lake Eyre dragons, elevated testosterone levels corre- sonal androgen proWles and potentially also demonstrate sponded with cessation of appeasement behaviours and that females exhibit some capacity for behavioural andro- mean progesterone levels were signiWcantly correlated with gen responses. For the latter we could simply measure the duration of rejection behaviours. There was no general plasma testosterone in females before and after bouts of correlation, however, between testosterone levels and dura- mate rejection behaviour. tion of rejection behaviours because during the period of Elevated levels of testosterone in both sexes are thought receptivity (stages 1–3), testosterone levels were very high to be associated with potential Wtness costs including (the Wrst peak) while rejection behaviours were absent or decreased parental care, reduced fecundity and suppressed rare. This does not exclude the possibility that testosterone immunocompetence (e.g. Fite et al. 2005; Folstad and could mediate rejection behaviour, particularly given that Karter 1992; Rutowska et al. 2005; Searcy 1988). While testosterone levels remained elevated (and even exhibited a some of these costs are irrelevant for Lake Eyre dragons second peak) during periods when females exhibited maxi- (e.g. parental care), any potential direct costs of testoster- mal courtship rejection (stages 4–6). one may be negligible relative to the costs of forced male More broadly in female vertebrates, the potential media- copulation, which include potential injury, death and tion of female mate rejection or aggressive behaviours by reduced oVspring Wtness (Olsson 1995a). Consequently, sex steroids, particularly testosterone, is now documented female Lake Eyre dragons may maintain elevated plasma in multiple lizards, birds and Wsh (Desjardins et al. 2006; testosterone, despite associated costs, in order to increase 123 J Comp Physiol A male rejection behaviours post-ovulation because these Amundsen T (2000) Why are female birds ornamented? TREE behaviours reduce the frequency of costly forced copula- 15:149–155 Amundsen T, Forsgren E (2001) Male mate choice selects for female tions (Chan et al. submitted). coloration in a Wsh. Proc Natl Acad Sci USA 98:13155–13160 Other steroids, including oestradiol, are presumably Amundsen T, Parn H (2006) Female coloration: a review of functional important in regulating or co-regulating physiology, colour- and non-functional hypotheses. In: Hill GE, McGraw KJ (eds) ation and behaviour in female Lake Eyre dragons. For Bird coloration: function and evolution. Harvard University Press, Boston, pp 280–348 example, the regulation of sexual receptivity in lizards Arnqvist G, Rowe G (2005) Sexual conXict. Princeton University appears to be a synergistic interaction between oestradiol Press, Princeton and progesterone (McNicol and Crews 1979; Whittier and Baird TA (2004) Reproductive coloration in female collared lizards, Tokarz 1992). In Lake Eyre dragons receptivity inferred by Crotophytus collaris, stimulates courtship by males. Herpetolog- ica 60:337–348 maximal copulation frequency peaked during stages 1–3 Barbour HR, Archer MA, Hart NS, Dunlop SA, Beazley LD, Shand J whilst progesterone levels were still increasing and both (2002) Retinal characteristics of the ornate dragon lizard, Cteno- progesterone and testosterone levels remained high when phorus ornatus. J Comp Neurol 450:334–344 females were no longer receptive. Consequently, for Lake Berger J (1983) Induced abortion and social factors in wild horses. Nature 303:59–61 Eyre dragons’ female receptivity was poorly correlated Calisi RM, Hews DK (2007) Steroid correlates of multiple color traits with plasma steroid proWles suggesting that other in the spiny lizard, Sceloporus pyrocephalus. J Comp Physiol B hormones, such as oestradiol, could be mediating this 177:641–654 behaviour in Lake Eyre dragons. 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Anim Behav 35:1177–1187 one and progesterone and associated changes in colouration Cooper WE, Crews D (1988) Sexual coloration, plasma concentrations and behaviour appear similar to those reported for North of sex steroid hormones, and responses to courtship in the female American Iguanid lizards with similar colour and keeled earless lizard (Holbrookia propinqua). Horm Behav 22:12–25 behavioural traits, including female Holbrookia propinqua Cooper WE, Ferguson GW (1972a) Relative eVectiveness of proges- (Cooper and Crews 1987). This concordance suggests a terone and testosterone as inductors of orange spotting in female potentially conserved regulatory role for these steroids in collared lizards. Herpetologica 28:64–65 mediating these reproductive traits in female lizards. Fur- Cooper WE, Ferguson GW (1972b) Steroids and color change during gravidity in the lizard, Crotaphytus collaris. 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