Abstract Superb fairy-wrens are cooperatively breedingbirds that combine stable, socially monogamous pairbonds and high levels of paternal care, with extreme lev-els of extra-pair mating and high levels of sexual compe-tition. Our aim was to determine which testosterone cor-relates would prevail in such a life history that combinesfeatures that are conventionally associated with diver-gent hormone profiles. Unlike the situation in other spe-cies with monogamous pair bonds and high levels of pa-ternal care, testosterone was elevated for a very long pe-riod of several months. During breeding there was abroad peak in testosterone followed by a gradual decline:this resembles the profile found in polygynous and pro-miscuous species. We found that three factors correlatedwith testosterone: development of the sexually selectednuptial plumage, social status and extra-group matingopportunities. Testosterone started increasing monthsprior to breeding, when the males that are later preferredas extra-group sires develop their nuptial plumage. Al-though these males did not have higher testosterone lev-els during breeding, they sustained high testosterone formuch longer, and this might lend reliability to this sexualsignal. Dominant males in groups had higher testoster-one than pair-dwelling males and subordinate helpers.This was not due to differences in age, reproductive ca-pability or mating opportunities, but was presumably as-sociated with the assertion of dominance. In contrast tofindings in other species, male testosterone level was notcorrelated with whether the resident female was fertile orhad dependent nestlings. However, testosterone wasstrongly correlated with the total number of fertile fe-

males in the population, and hence with the opportunitiesfor extra-group mating.

Keywords Testosterone · Sexual selection · Cooperativebreeding · Extra-pair mating

Introduction

Trade-offs in vertebrate life histories are likely to be hor-monally mediated since hormones typically influencemany behaviours simultaneously (Stearns 1989). Inbirds, the steroid hormone testosterone has receivedmuch attention because it affects a wide range of behavi-ours central to male life history trade-offs (Ketterson etal. 1996). Low levels of testosterone suffice for the de-velopment of primary and secondary sexual characteris-tics. High testosterone levels play a pivotal role in socialand sexual interactions, for example stimulating aggres-sive, mate attraction and mate-guarding behaviours andsuppressing paternal care (Ketterson and Nolan 1994).

In free-living birds, the temporal pattern and ampli-tude of elevated testosterone vary between mating sys-tems (Wingfield et al. 1990). Specifically, testosteronelevels reflect the degree of male-male aggression andmale contribution to offspring care: as competition de-creases and paternal care increases the period of hightestosterone becomes shorter and the peak lower. Ac-cordingly, males in socially monogamous species withhigh levels of paternal care typically have a short, dis-tinct peak in testosterone when the female is fertile, afterwhich it returns to basal levels. Conversely, polygynousmales that provide little care have higher levels of testos-terone for a longer period (Wingfield 1990).

Patterns of testosterone not only reflect the essence ofthe mating system but also the social system. For exam-ple, in cooperatively breeding species, testosterone lev-els may vary in males of different social status. Subordi-nate male helpers are often reproductively less activecompared to dominant males and they may show lowerlevels of plasma steroids and smaller gonads (Reyer et

Communicated by J. Dickinson

A. Peters (✉ ) · A. CockburnDivision of Botany and Zoology, Australian National University,Canberra, ACT 0200, Australiae-mail: anne.peters@anu.edu.auTel.: +61-2-61252866, Fax: +61-2-61255573

L.B. AstheimerDepartment of Biomedical Science, Northfields Avenue, University of Wollongong, NSW 2522, Australia

Behav Ecol Sociobiol (2001) 50:519–527DOI 10.1007/s002650100403

O R I G I N A L A RT I C L E

Anne Peters · Lee B. Astheimer · Andrew Cockburn

The annual testosterone profile in cooperatively breeding superb fairy-wrens, Malurus cyaneus, reflects their extreme infidelity

Received: 31 January 2001 / Revised: 25 June 2001 / Accepted: 11 July 2001 / Published online: 17 August 2001© Springer-Verlag 2001

al. 1986; Schmidt et al. 1991; Schoech et al. 1991;Poiani and Fletcher 1994). However, the extent to whichlower testosterone levels in helpers are merely correlatedwith their younger age, sexual immaturity or lack ofbreeding opportunities rather than being due to their sub-ordinate status per se remains unclear (but see Reyer etal. 1986).

To date, most studies of the role of testosterone inavian life histories have used model systems that exhibiteither monogamy with high paternal care or polygynywith low paternal care, in either cooperative or pair-breeding systems. In contrast, superb fairy-wrens, Mal-urus cyaneus, provide the opportunity to examine thetestosterone profile in a species that exhibits features thatare conventionally associated with divergent hormonepatterns.

Superb fairy-wrens are cooperative breeders thatcombine a monogamous social system and high paternalcare with a mating system based on extra-group fertilisa-tion and intense sexual selection. Fairy-wrens live aspairs that can be assisted in raising their offspring by upto four behaviourally subordinate but reproductivelycompetent male helpers. Males are seasonally dichro-matic and all males develop a colourful nuptial plumagein their first breeding season (Mulder et al. 1994). Most(76%) offspring in the population are sired by malesfrom outside their social group and the successful extra-group sires are those that develop their colourful plum-age long before the start of breeding (Dunn andCockburn 1999). While in nuptial plumage, all malesfrequently visit extra-group females to perform courtshipdisplays (Mulder 1997). Despite consistently high levelsof extra-group courtship and mating, males contributesubstantially to offspring care, providing up to 50% ofnestling feeds (Green et al. 1995; Dunn and Cockburn1996).

We studied the annual pattern of testosterone levels inrelation to the social and mating system in superb fairy-wrens. We aimed to establish whether this pattern corre-lates with either the behavioural or the genetic matingsystem, that is, whether the testosterone profile resem-bles that of a monogamous, biparental species or a poly-gynous species with continued high investment in mat-ing. We determined seasonal testosterone levels in maleswith and without paternal duties while within- and extra-group mating opportunities varied. In addition, we aimedto establish if male testosterone levels were correlatedwith their attractiveness to extra-group females, mea-sured as the date of the moult into colourful nuptialplumage. Finally, we investigated the relationship be-tween male social status and testosterone level by com-paring similar-aged males living in pairs as dominant orsubordinate males in groups. Our findings suggest thatthe testosterone profile is largely determined by the ac-quisition of the nuptial plumage and the mating system,while subtle differences in testosterone levels are relatedto male social status.

Methods

Study species

Superb fairy-wrens are small (9–10 g), long-lived (up to 11 years),cooperatively breeding passerines. They occupy year-round all-purpose territories as socially monogamous pairs which in 60% ofcases are assisted by one to four helper males. In groups, the se-nior male is behaviourally dominant over the younger, subordinatehelpers, and dominance is usually achieved by orderly queuing:when the dominant male dies, the next-oldest helper male auto-matically becomes dominant. Regardless of social status, all malesare capable of breeding at 1 year of age when they develop a full-sized cloacal protuberance (see below; Mulder and Cockburn1993), the ability to fertilise eggs (Dunn and Cockburn 1999) anda colourful nuptial plumage (Mulder and Magrath 1994).

Male plumage alternates between dull brown eclipse plumageand iridescent blue-and-black nuptial plumage. Whereas malesmoult synchronously into eclipse plumage in late summer, theyinitiate moult into nuptial plumage at any time from early autumnuntil late spring (March until November; Dunn and Cockburn1999). By the end of spring, all males have developed a completenuptial plumage, and there is little variability in the extent or in-tensity of the plumage attained (A. Cockburn, unpublished data).In spring, all males also develop a cloacal protuberance, a largebulbous swelling that contains the seminal glomera, the site ofsperm storage (Mulder and Cockburn 1993).

Early acquisition of the nuptial plumage is an age- and condi-tion-dependent indicator of male quality (Dunn and Cockburn1999; Peters 2000): males moult progressively earlier until theyare 5 years old, whereas they delay moult in harsh winters(Mulder and Magrath 1994). Testosterone levels rise during themoult, and males in blue plumage always have elevated testoster-one, even in mid-winter, despite an immune suppressive effect oftestosterone (Peters 2000; Peters et al. 2000). Individual malesshow high consistency in relative moult date between years(Mulder and Magrath 1994). Male reproductive success is heavilyskewed: males that complete the nuptial plumage at least 1 monthprior to the start of the breeding season are strongly preferred asextra-group mates (Dunn and Cockburn 1999).

Despite year-round territoriality and social stability, extra-group matings are extraordinarily common (93% of nests, 76% ofoffspring; Mulder et al. 1994). Females are in control of extra-group mating (Double and Cockburn 2000) and males court fe-males intensely: males of all ages and social classes engage in fre-quent (average 15–20 per day; Green et al. 1995) courtship dis-plays directed exclusively at extra-group females (Mulder 1997).When nestlings require care, dominant males can maintain a high-er rate of courtship displays because helpers compensate for lossof nestling care (Green et al. 1995). However, this comes at a cost:helpers also liberate females from constraints on extra-group matechoice, and dominant males in groups lose more paternity to extra-group sires than do males in unassisted pairs (Mulder et al. 1994).

Study population

All males for this study were taken from a colour-banded popula-tion in and around the Australian National Botanic Gardens(ANBG) Canberra, Australia. The ANBG is a reserve of dry scle-rophyll forest and irrigated plantations of Australian native flora.Superb fairy-wrens have been studied at the ANBG since 1988and most birds are of known age. Between 1996 and 1998, ap-proximately 90 breeding groups were monitored each year. Groupcomposition and dominance status of resident males were sur-veyed year-round for all social groups. During the breeding sea-son, initiation and progress of the breeding activities of all femaleswere followed closely in daily censuses. During the non-breedingseason, all males were monitored for signs of initiation, progressand completion of the pre-nuptial moult (for a detailed descriptionsee Dunn and Cockburn 1999). Although females may initiate upto nine clutches per season, nest predation is high such that they

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fledge at most three broods per year (A. Cockburn, unpublisheddata).

Plasma sampling

Birds were captured in mist nets between 0550–1130 hours (medi-an 0730 hours) and weighed to the nearest 0.1 g using a 30-gPesola spring balance. We measured head-bill and tarsus length aswell as the length, width and depth of the cloacal swelling(Mulder and Cockburn 1993) to the nearest 0.05 mm using Verniercalipers. We estimated the percentage of visible coloured nuptialfeathers (Dunn and Cockburn 1999). A small (60–140 µl) bloodsample was collected from the brachial vein into heparinised cap-illary tubes. Tubes were stored vertically on ice until they weretransported to the laboratory (within 5 h) and spun in a microhae-matocrit centrifuge for 3 min at 12,000 rpm. Haematocrit wasmeasured and the plasma separated from the packed cells andstored at –70 C. Blood samples were taken as soon as possible af-ter capture (median=20 min, mean=25 min, SE=0.8, range5–85 min). Because testosterone levels can vary during the dayand can be affected by long delays between capture and bloodsampling (Wingfield et al. 1982), we initially included the time ofcapture and the delay to sampling in all statistical analyses. Therewas no evidence for an effect of time of day or of a linear or non-linear effect of the delay between capture and sampling on testos-terone levels (all P>0.25).

Testosterone analysis

Plasma testosterone concentrations were determined within10 months of plasma collection using a commercial radioimmuno-assay (Pantex, Santa Monica, Calif.). This assay shows high speci-ficity for testosterone and 7.9–10.5% intra-assay and 8.1–12% in-ter-assay variability. We modified this assay to accommodatesmall plasma volumes and low testosterone titres by halving thevolumes of all other assay reagents, thus effectively doubling therecommended sample volume and increasing assay sensitivity.Plasma samples were analysed in two replicates of 10–30 µl eachand when replicate values differed by more than 10%, the sampleswere excluded from analysis. The effective range of the assay wasconservatively truncated below 0.10 ng/ml (approximately 90%binding) and testosterone values below the assay detection limitwere set at 0.10 ng/ml accordingly (Peters et al. 2000).

Statistical analysis

The superb fairy-wren breeding cycle consists of three phases thatare distinct in terms of male moult, female breeding activity and

male testosterone levels: pre-breeding (1 June–31 September);breeding (1 October–14 January) and post-breeding (15 January–31 May; Table 1). Testosterone levels during the post-breedingphase showed negligible variation. Since the temporal testosteronepattern differed substantially during the pre-breeding and breedingphase (Fig. 1), we developed separate multiple-regression modelsfor these phases to determine which variables best explained thetestosterone profile (see below for details).

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Table 1 Testosterone levels, mating opportunities and male plumage status during the three phases of the superb fairy-wren breedingcycle

Phase Number of Testosterone Number of Plumagemales captured (ng/ml) fertile femalesa

Mean (SD, Mean (SD, Brown Moult Bluemaximum) maximum)

Pre-breeding 125 0.30 (0.35, 2.4) 1 (2, 9)b 57 20 48(1 June–31 September)

Breeding 68 0.45 (0.63, 3.3) 14 (6, 29) 0 1 67(1 October–14 January)

Post-breeding 36 0.11 (0.05, 0.3) 0 (0, 0) 23 12 1(15 January–31 May)

a Daily total of all females in the population in the fertile phase(between 6 days before the start of laying and the day of the pen-ultimate egg)

b A small number of females initiated breeding exceptionally earlyin 1998; in 1996 and in 1997, no female became fertile before 29 October

Fig. 1 Annual variation in testosterone levels in male superbfairy-wrens (a) and the number of males captured in blue plumageand number of fertile females in the population (b). Males werecaptured between February 1996 and September 1998. Testoster-one levels and proportion of blue males are monthly means±SE ofall males sampled during the study; numbers refer to sample sizes.Number of fertile females are monthly means±SE of the daily to-tal number of females (out of 85–90) in the population that werebetween 6 days before the start of laying and the day of the penul-timate egg

For all analyses, testosterone concentrations were transformedby natural logarithm to fit standard least-squares models. We useda top-down approach in all multiple regression models, initiallyincluding all explanatory variables (see below for details) and pro-gressively eliminating non-significant terms (P>0.05). All elimi-nated variables were reintroduced to the final model to confirmthe lack of contribution, and statistical details presented (F, dfs,P-values) are based on the change in variance resulting from addi-tion of the term to the final model. Residual plots and normalprobability plots were examined for unequal variance and devia-tions from normality among residuals to verify the validity of thefinal model. All results presented (means±SE; back-transformedwhere appropriate) are predicted by a model containing all signifi-cant terms. Statistical analyses were calculated using Genstat 4.5.1(Genstat 1997).

Pre-breeding phase multiple regression model

Males were sampled in 1996, 1997 and 1998. Some males(19/105) were represented more than once. However, most ofthese were sampled in different years (mean±SE number of daysbetween captures: 327±70). In addition, a separate analysis of 13 males that had been sampled twice within 6 weeks (from a totalof 167 males sampled across the three periods) revealed no evi-dence for a correlation between repeated samples (A. Peters, un-published data). In view of the lack of evidence for dependenceand the small number and the long time span between repeatedsamples, we treated samples as independent.

Before examining the significance of any parameters, we de-termined which function of sampling date and year best summari-sed the temporal pattern of testosterone. The model was derivedthrough an iterative process by first fitting a curve to the relation-ship between testosterone and date for each year separately. Ini-tially, we fitted separate fourth-order smoothing functions to thedate, one for each year. We then progressively reduced the com-plexity of the functions until further reduction resulted in a signifi-cant (P<0.05) difference in variance ratio between the more com-plex and the reduced model. After we had thus established thebest-fitting description of the temporal testosterone pattern, wesubsequently tested for the additional contributions of male moultstatus (blue, in moult, brown), presence of any cloacal swelling(yes/no), male age (1, 2, 3 and 4 or older), haematocrit and malebody condition (estimated as the residual of a regression of bodymass at capture on the first principal component of tarsus and headlength).

Breeding-phase multiple regression model

Males were sampled in 1996 and 1997 and all males were repre-sented only once in the sample. Because one of the aims was todetermine the correlation of testosterone and social status, we in-cluded only one male from a social group (if we had sampled sev-eral males in a group we chose one at random). As a measure formating opportunity, we calculated the number of fertile females inthe population on a given day as the sum of all females that werebetween 6 days before the start of laying and the day of the penul-timate egg. This 9- to 10-day period includes the peaks in bothmale courtship display (Mulder 1997; Green et al. 2000) and fe-male sexual behaviour (Double and Cockburn 2000). As a mea-sure for male phenotypic quality, we calculated the within-age-co-hort rank for the date of acquisition of the nuptial plumage in theyear the male was captured (Mulder and Magrath 1994). For 1996and 1997 separately, we ranked all males of 1, 2–3 and 4 or olderby date of moult completion and calculated a weighted rank scorefor each male (JMP v. 3.0.2, SAS Institute), with the first moultingmale(s) given a score of 1 and the last moulting male(s) a score of0; these scores were then used as an indicator of male phenotypicquality. Volume of the cloacal protuberance was calculated as πlr2

with l=length of the protuberance and r=1/4(width+height) (Mul-der and Cockburn 1993). After we had determined which function

of sampling date and year best summarised the temporal pattern oftestosterone, as described above for the pre-breeding season, weconstructed full models containing year, date, male social status(dominant in group, subordinate in group, in pair), fertility statusof the resident female (fertile yes/no), presence of dependentyoung (yes/no), number of fertile females in the population, vol-ume of the cloacal protuberance, male age (1, 2, 3 and 4 or older),male body condition (estimated as the residual of a regression ofbody mass at capture on the first principal component of tarsusand head length) and male phenotypic quality (within-age-cohortrank of moult date; see above). Apart from these main effects, wealso included single-order interaction terms between male socialstatus and female fertility status, presence of dependent young andnumber of fertile females.

Results

We distinguished three phases during the superb fairy-wren breeding season (Table 1). These are distinct interms of male plumage status, female breeding activityand male testosterone levels and we analysed them sepa-rately.

Pre-breeding

From the start of winter, progressively more males ofhigh quality initiate the pre-nuptial moult. Consequently,during the pre-breeding phase, the number of males innuptial plumage gradually increases, even though severalmonths may elapse before females initiate breeding(Fig. 1b).

Testosterone levels gradually increase during the pre-breeding phase (Fig. 1a), with no evidence for non-linearity (F1,120=1.5, P=0.2). Analysis of the temporalpattern of testosterone during this period revealed aweak interaction between year and date (F2,118=3.1,P=0.05). This was because testosterone levels increasedto peak levels earlier in 1998 due to unusually earlybreeding in that year. In 1998, females initiated repro-duction earlier than in any other year (A. Cockburn, un-published data 1988–1998; see also Peters et al. 2000);in 1996 and 1997 no female was fertile during the pre-breeding period (Table 1). Apart from this interaction,there was a significant effect of male plumage state (inblue plumage, in moult or in brown plumage; F2,118=33,P<0.001). The increase of testosterone levels did notcorrelate significantly with the development of the clo-acal swelling (F1,117=3.4, P=0.066), age (F4,109=2.0,P=0.1), haematocrit (F1,116=0.1, P=0.7) or male bodycondition (F1,116=0.6, P=0.4). Thus, apart from a weakinteraction between year and date, in the period prior tobreeding, by far the most important correlate of testoster-one levels was development of the nuptial plumage. Theincrease in testosterone levels during the pre-breedingperiod (Fig. 1a) was associated with an increase in thenumber of males in blue plumage (Fig. 1b), and males inblue plumage always had higher testosterone levels(Fig. 2). While testosterone levels were always differentbetween the plumage groups, there was no change in tes-tosterone levels within each of these three groups overtime (Fig. 2).

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Breeding season

Initially, we investigated the temporal pattern of testos-terone including only date and year as explanatory vari-ables. Testosterone levels differed between the two sam-ple years (F1,62=6.0, P=0.017) and declined linearlythroughout the season (F1,62=6.3, P=0.015). There wasno evidence for non-linearity (F1,62=1.0, P=0.3) or an in-teraction between year and date (F1,62=1.5, P=0.2). How-ever, when we included additional explanatory variablesin the model, the evidence for effects of date and yearweakened, and neither date (F1,53=2.5, P=0.11), year(F1,53=0.15, P=0.7) nor their interaction term (F1,51=0.5,P=0.5) made a significant contribution. This was mostlikely due to the strong positive correlation between thenumber of fertile females in the population and male tes-tosterone levels (F1,54=27, P<0.001; Fig. 3). In addition,male social status influenced testosterone levels, withhighest levels in dominant males in groups (F2,55=5.4,P=0.007; Fig. 3). We found no evidence that the age

(F3,48=0.9, P=0.4, Fig. 4), relative moult quality(F1,41=2.6, P=0.12; Fig. 5) or body condition (F1,52=0.2,P=0.7) of a male were significantly correlated with histestosterone levels. Male testosterone levels were not as-sociated with the fertility status of the resident female(F1,53=0.09, P=0.7; Fig. 6a) or the presence of dependentnestlings (F1,53=0.1, P=0.7; Fig. 6b). There was no evi-dence that interactions between male social status andthe presence of dependent nestlings (F2,52=0.5, P=0.6) orbetween the two significant main effects, male social sta-tus and the number of fertile females in the population

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Fig. 2 Testosterone levels during the pre-breeding season in malesin brown plumage, in pre-nuptial moult and in blue nuptial plum-age. Males were captured between 1 June and 31 September in1996, 1997 and 1998. Symbols are the observed data; the lines arepredicted by a multiple regression model

Fig. 3 The correlation between the number of fertile females inthe population and male testosterone levels (natural log-trans-formed) for dominant and helper males in groups and males inpairs. Males were captured during two breeding seasons, between1 October and 15 January in 1996 and 1997. Symbols are the ob-served data; the lines are predicted by a multiple regression model

Fig. 4 Testosterone levels in males of 1, 2 or 3, and 4 or moreyears of age captured during two breeding seasons, between 1 Oc-tober and 15 January in 1996 and 1997. Shown are back-trans-formed means±SE, predicted by a multiple regression model con-trolling for male social status and the number of fertile females inthe population; numbers in bars refer to sample sizes

Fig. 5 Male phenotypic quality and testosterone levels (naturallog-transformed) in males captured during two breeding seasons,between 1 October and 15 January in 1996 and 1997. Male qualityis calculated as a weighted, within-age-cohort rank of date of com-pletion of the nuptial plumage in the preceding spring; malesranked as 1 were the first of their age group to develop the nuptialplumage in the year of study, and males ranked as 0 were the last

(F2,53=0.9, P=0.4), made a significant contribution to thefinal model (Fig. 3).

Post-breeding

During the post-breeding season, testosterone levelswere basal and breeding activity declined (Fig. 1). Afterlate summer (mid-January), no new nesting attemptswere initiated, although females continued incubatingand adults cared for nestlings and then fledglings untillate March. In late summer and early autumn, males andfemales underwent the post-breeding moult, males de-veloping their brown eclipse plumage. Typically, only asmall number of exceptional males developed the nuptialplumage during autumn. In 1996 and 1997, in total only11 of 451 males were in blue plumage between 15 Janu-ary and 31 May and consequently we captured just one

blue male during the post-breeding period. Accordingly,testosterone levels were basal throughout (Table 1): in95% (34 of 36) of males, testosterone was below the levelof detection.

Discussion

The seasonal pattern of testosterone levels in superbfairy-wren males reflects a long breeding season preced-ed by a long period of increased sexual signalling. Ele-vated levels of testosterone occurred over a 7-month pe-riod, from June until January. From early winter, testos-terone levels slowly rise as increasing numbers of malesdevelop their blue nuptial plumage. Testosterone reachesmaximum values in the middle of spring, followed by agradual decline until mid-summer (mid-January; Fig. 1).This pattern is different from that observed in other mo-nogamous birds with biparental care, where testosteronetypically shows a brief peak, for a few days to weeks,during nest building and egg-laying, followed by a sharpdecline at the onset of paternal behaviours (for a reviewsee Wingfield et al. 1990). Similar prolonged elevationof testosterone during most of the breeding season hasbeen observed in polygynous red-winged blackbirds,Agelaius phoeniceus, that do not provide paternal care.In this species, testosterone is elevated for a 5-week pla-teau (Beletsky et al. 1989). The fairy-wren testosteroneprofile during breeding also closely resembles that of thebrown-headed cowbird, Molothrus ater. Cowbirds areobligate brood parasites that provide no parental care,while females lay eggs continuously throughout thebreeding season. As in fairy-wrens, testosterone reachesmaximum values in the middle of spring followed by agradual decline until mid-summer (Dufty and Wingfield1986; Fig. 1a). The lack of similarity with the relativelybrief spike in testosterone levels commonly observed inmonogamously breeding species with high paternal careshows that the testosterone profile in superb fairy-wrensis not governed by the principles that typically prevail insuch species.

Although the duration of elevated testosterone is pro-longed in fairy-wrens, the amplitude of the increase intestosterone is relatively modest, especially compared tomigrants from the temperate zone of the northern hemi-sphere. These birds show absolute peak testosterone lev-els about ten times higher than in fairy-wrens (for exam-ple Wingfield and Farner 1979; Hunt et al. 1995). Theseextremes however, are observed for a short time only,and are associated with periods of intense aggression(Wingfield et al. 1990). In species that are social andmaintain long-term pair bonds, testosterone levels, evenwhen elevated, are relatively low (Reyer et al. 1986;Mays et al. 1991; Wingfield et al. 1991). Moreover, inold Australian endemics – that show a propensity for co-operative breeding (Cockburn 1996) – peak testosteronelevels may be reduced in amplitude (Schmidt et al. 1991;Poiani and Fletcher 1994; McDonald et al. 2001). Ex-treme peak values of testosterone would not be expected

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Fig. 6 Testosterone levels in relation to breeding phase: maleswith a fertile resident female compared to males where the resi-dent female was not fertilisable (a) and males caring for depen-dent nestlings and males without dependent young (b). Maleswere captured between 1 October and 15 January in 1996 and1997. Shown are back-transformed means±SE, predicted by amultiple regression model including male social status and thenumber of fertile females in the population; numbers in bars referto sample sizes

in fairy-wrens, because large changes in testosterone arelikely to result in very deterministic, inflexible responsesthat would be inappropriate for such a sophisticatedsystem of long-term social interactions and intricate rela-tionships between resident individuals. Modest fluctua-tions in testosterone allow scope for fine-tuned adjust-ment of individual behaviour.

In accord with their unusual mating system, we foundthat three factors are of key importance in explaining in-dividual testosterone levels in male superb fairy-wrens:development of the sexually selected nuptial plumage,extra-group mating opportunities and social status.

Sexual signalling

Testosterone levels start rising long before breedingstarts, coincident with the onset of sexual advertisementduring the pre-breeding phase (Fig. 1b). This increase isa result of increasing numbers of males that have ac-quired the blue plumage: testosterone levels in blue,brown or moulting males do not increase during this pe-riod (Fig. 2). This is entirely consistent with our findingthat testosterone increases during the moult and remainselevated in males in blue plumage, even in mid-winter(Peters et al. 2000).

In recent years, there has been a surge of interest in therole of testosterone in the production of sexual ornamentsand signals. Testosterone is proposed to act as a physio-logical link between the level of sexual signalling andmale condition, with higher-quality males having highertestosterone and more exaggerated sexual signals (Folstadand Karter 1992). In general, plumage ornaments are con-sidered less effective at signalling individual testosteronelevels than testosterone-dependent behaviours or fleshystructures such as wattles and combs (Zuk et al. 1995). Incontrast to these flexible traits whose maintenance re-quires a continuous level of testosterone (Zuk et al. 1995;Eens et al. 2000), fixed plumage traits do not necessarilycorrelate with testosterone levels after the moult is com-pleted. This appears to be the case in fairy-wrens: indi-vidual testosterone levels during the breeding phase werenot correlated with the time when the male had developedthe nuptial plumage (Fig. 5). Hence, in fairy-wrens, thesexually selected signal of male quality, the early moultinto nuptial plumage, does not indicate individual testos-terone level during breeding.

However, early acquisition of the nuptial plumage ac-curately signals the duration of elevated testosterone:high-quality males, which produce the blue plumage be-fore mid-winter, sustain high levels of testosterone formost of the year. This, in conjunction with Peters' (2000)finding that testosterone is immunosuppressive, supportsthe hypothesis by Dunn and Cockburn (1999) that theearly moult is a signal of endurance guaranteeing thehonesty of the signal. In fairy-wrens, females may disre-gard instantaneous male display rate (Green et al. 2000)and individual testosterone during the breeding period(Fig. 5) and select extra-group mates on the basis of pro-longed maintenance of blue plumage and associated ele-

vated testosterone levels, which are more likely to behonest indicators of quality (Fig. 2; Dunn and Cockburn1999; Green et al. 2000; Peters et al. 2000).

Extra-group mating opportunities

Contrary to what is expected for birds with biparentalcare, male testosterone levels were not correlated withthe breeding stage on the home territory. First, testoster-one levels did not decline when males were provisioningnestlings (Fig. 6b). One of the most universal hormone-behaviour relationships in vertebrates is the negative cor-relation between testosterone and paternal care: testos-terone suppresses parental behaviours and it is alwaysdepressed when young receive care (Wingfield andMoore 1987; Wingfield et al. 1990; Ball 1991; Vleckand Brown 1999). As far as we know, fairy-wrens are theonly species with elevated testosterone in males that arecaring for dependent young – all males care for nes-tlings, and the level of care is not correlated with the tes-tosterone level (A. Peters and C. Cockburn, unpublisheddata). Second, testosterone levels in male fairy-wrens didnot vary with the fertility status of their mate (Fig. 6a),although they share the same territory year-round. Gen-erally, males are highly sensitive to the fertility status oftheir mate, and show an abrupt, steep rise in testosteronelevel when their mate is fertile (Wingfield et al. 1990).

In contrast to the absence of a correlation between themales' testosterone level and the fertility status of theirown mate, there was a strong positive correlation withthe total number of fertile females in the population(Fig. 3). Initial statistical modelling of the seasonal trendduring the breeding season revealed that testosteronelevels differed between years, and varied with the date.However, when the daily mean number of fertile femaleswas included in the model, the statistical effects of dateand year on testosterone levels disappeared. This indi-cates that the correlations of testosterone with year anddate were a result of annual and seasonal fluctuations inthe number of fertile females in the population.

That testosterone can increase as a response to thepresence of a receptive female has been well established.Numerous studies have reported a rise in male testoster-one levels in monogamous, polygynous and cooperativelybreeding males when a mate initiates a clutch (Beletskyet al. 1989; Wingfield et al. 1990; Mays et al. 1991;Schoech et al. 1996). In polygynous red-winged black-birds, the presence of an additional receptive female latein the season causes elevations in male testosterone lev-els (Johnsen 1998). Likewise, in Mexican jays, Aphelo-coma ultramarina, where there are several breeding fe-males in a group, some females were still fertile whenothers were incubating and, consequently, the periodduring which males had elevated testosterone was pro-longed (Vleck and Brown 1999). Ours is the first reportof an association between individual male testosteronelevels and the number of fertile females in the popula-tion, and this is most likely linked to the mating systemand the intense female choice for extra-group paternity.

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The opportunities for extra-group matings, which giverise to the majority of offspring (Mulder et al. 1994), aredirectly correlated with the number of fertile females: fe-males only mate with extra-group males at the peak oftheir fertility (Double and Cockburn 2000). Male fairy-wrens are aware of the fertility status of females: theyare regularly seen inspecting nests (unpublished data)and the number of extra-group courtship displays direct-ed at a female increases when she is fertile (Green et al.2000). Thus, a rise in the potential for extra-group mat-ing could stimulate a rise in testosterone in individualmales. Likewise, we would predict a correlation betweenmale testosterone levels and the number of fertile fe-males in other species with high and variable opportuni-ties for extra-pair mating.

Reproductive suppression or a cost of helpers?

Testosterone varied significantly with male social status:testosterone levels of dominant males in groups werehigher than those of males in pairs and helpers in groups.Males without helpers had comparable levels to helpersin groups (Fig. 3). This variation in testosterone betweenmales of different social status in fairy-wrens was not aresult of differences in age (Fig. 4). In many social andcooperatively breeding species, helpers, or non-breeders,have low testosterone during breeding. These helpers arepotentially capable of breeding, but their gonads are in-completely developed and their testosterone levels sup-pressed (e.g. bell miners, Manorina melanophrys: Poianiand Fletcher 1994; Florida scrub jays, Aphelocoma coe-rulescens: Schoech et al. 1991), although this is not thecase for those helpers that have substantial mating op-portunities (e.g. pied kingfishers, Ceryle rudis: Reyer etal. 1986; Harris' hawks, Parabuteo unicinctus: Mays etal. 1991). In superb fairy-wrens, all males are sexuallymature, develop a complete nuptial plumage (Mulderand Magrath 1994), a full-sized cloacal protuberance(Mulder and Cockburn 1993), and are capable of fertili-sing eggs (Dunn and Cockburn 1999). Therefore, weconclude that the difference in testosterone level be-tween dominant males in groups, males in pairs andhelpers in groups is not a result of a difference in repro-ductive capability.

Lower testosterone levels in helpers/non-breedershave often been interpreted as reproductive suppressionby the dominant male (Reyer et al. 1986; Schoech et al.1991; Wingfield et al. 1991; Vleck and Brown 1999). Infairy-wrens, testosterone is apparently not lowered inhelpers, as testosterone in helpers is comparable to thatin males in pairs, but, instead, testosterone is higher indominant males (Fig. 3). This could be linked to the ex-pression of dominance. Testosterone levels can rise as aresult of aggressive or sexual interactions (Wingfield1990). In Florida scrub jays, testosterone levels in breed-ers were higher when more non-breeding males werepresent in the population. Schoech et al. (1996) attribut-ed this to a higher incidence of aggressive interactionsbetween males. Likewise, dominant male fairy-wrens

may have elevated testosterone as a result of assertingtheir dominant status.

In superb fairy-wrens, dominance status has somebenefits but also considerable costs. Dominant males ac-tively punish helpers for defection from group activities.When helpers were temporarily removed when depen-dent nestlings were present, dominants did not adjust pa-ternal investment, but violently attacked the helperswhen they were returned to the group (Mulder and Lang-more 1993). The principal benefit of dominance status isthat dominant males can invest less in nestling feeding,and more in courtship displays while their helpers makeup for their lower contribution to provisioning (Green etal. 1995). However, because helpers liberate femalesfrom constraints on extra-group mate choice, dominantmales lose more paternity to extra-group sires than domales in pairs (95% compared to 50%; Mulder et al.1994). Elevated testosterone levels in dominant malesmight constitute an additional cost of dominance: testos-terone is immune suppressive in fairy-wrens, at least inwinter (Peters 2000), and if high testosterone increasesthe risk of immune suppression during the breeding sea-son, this would constitute an additional cost of domi-nance. Generally, the hormonal basis of cooperativebreeding is discussed in terms of costs to helpers of re-duced testosterone, but our study shows that costs todominant males should also be considered.

Conclusion

The testosterone profile in superb fairy-wrens reflectsthe unusual mating system with extremely high levels ofextra-group paternity driven by intense sexual selectionand female choice. As in many species that are socialand maintain long-term pair bonds, peak testosteronelevels are relatively modest, allowing for fine-tuned reg-ulation of subtle behaviours in accordance with long-term intricate social relationships. However, the periodover which testosterone is elevated is very long, particu-larly in those males that are strongly preferred by fe-males, because they develop the colourful nuptial plum-age many months prior to the start of breeding. Duringbreeding, dominant males in groups have higher testos-terone, not due to differences in age, reproductive matu-rity or mating opportunities, but presumably as a resultof dominance interactions. Most surprisingly, testoster-one levels are not correlated with either the fertility ofthe resident female or the presence of dependent nes-tlings in the group, despite social monogamy and highlevels of paternal care. Individual male testosterone lev-els, however, are sensitive to the opportunities for extra-group matings, that is, the number of fertile females inthe population. Taken together, these findings indicatethat the testosterone profile in superb fairy-wrens is gov-erned by those aspects of the mating system directly cor-related with male fitness.

Acknowledgements The expert statistical help of Ross Cunning-ham and Christine Donnelly greatly facilitated analysis of thisstudy. We are grateful to the many people who provided help cap-

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turing the birds. Fieldwork was performed with permission fromthe Australian Capital Territory Parks and Conservation Service(licences LT97070 and LT98034 to A.P.), the ABBBS and theAustralian National Botanic Gardens (permit number 23, to A.C.).All protocols were approved by the Animal Experimentation Eth-ics Committee of the Australian National University, protocolnumbers F.BTZ.27.93 and F.BTZ.62.96. Research was supportedby APA/OPR Scholarships and a Cayley memorial scholarship toA.P. and an ARC grant to A.C. All research complied with animalexperimentation and quarantine laws of Australia.

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