Behav Ecol Sociobiol (2001) 50:519–527 DOI 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
Abstract Superb fairy-wrens are cooperatively breeding birds that combine stable, socially monogamous pair bonds and high levels of paternal care, with extreme levels of extra-pair mating and high levels of sexual competition. Our aim was to determine which testosterone correlates would prevail in such a life history that combines features that are conventionally associated with divergent hormone profiles. Unlike the situation in other species with monogamous pair bonds and high levels of paternal care, testosterone was elevated for a very long period of several months. During breeding there was a broad peak in testosterone followed by a gradual decline: this resembles the profile found in polygynous and promiscuous species. We found that three factors correlated with testosterone: development of the sexually selected nuptial plumage, social status and extra-group mating opportunities. Testosterone started increasing months prior to breeding, when the males that are later preferred as extra-group sires develop their nuptial plumage. Although these males did not have higher testosterone levels during breeding, they sustained high testosterone for much longer, and this might lend reliability to this sexual signal. Dominant males in groups had higher testosterone than pair-dwelling males and subordinate helpers. This was not due to differences in age, reproductive capability or mating opportunities, but was presumably associated with the assertion of dominance. In contrast to findings in other species, male testosterone level was not correlated with whether the resident female was fertile or had dependent nestlings. However, testosterone was strongly correlated with the total number of fertile feCommunicated by J. Dickinson A. Peters (✉) · A. Cockburn Division of Botany and Zoology, Australian National University, Canberra, ACT 0200, Australia e-mail:
[email protected] Tel.: +61-2-61252866, Fax: +61-2-61255573 L.B. Astheimer Department of Biomedical Science, Northfields Avenue, University of Wollongong, NSW 2522, Australia
males in the population, and hence with the opportunities for extra-group mating. Keywords Testosterone · Sexual selection · Cooperative breeding · Extra-pair mating
Introduction Trade-offs in vertebrate life histories are likely to be hormonally mediated since hormones typically influence many behaviours simultaneously (Stearns 1989). In birds, the steroid hormone testosterone has received much attention because it affects a wide range of behaviours central to male life history trade-offs (Ketterson et al. 1996). Low levels of testosterone suffice for the development of primary and secondary sexual characteristics. High testosterone levels play a pivotal role in social and sexual interactions, for example stimulating aggressive, mate attraction and mate-guarding behaviours and suppressing paternal care (Ketterson and Nolan 1994). In free-living birds, the temporal pattern and amplitude of elevated testosterone vary between mating systems (Wingfield et al. 1990). Specifically, testosterone levels reflect the degree of male-male aggression and male contribution to offspring care: as competition decreases and paternal care increases the period of high testosterone becomes shorter and the peak lower. Accordingly, males in socially monogamous species with high levels of paternal care typically have a short, distinct peak in testosterone when the female is fertile, after which it returns to basal levels. Conversely, polygynous males that provide little care have higher levels of testosterone for a longer period (Wingfield 1990). Patterns of testosterone not only reflect the essence of the mating system but also the social system. For example, in cooperatively breeding species, testosterone levels may vary in males of different social status. Subordinate male helpers are often reproductively less active compared to dominant males and they may show lower levels of plasma steroids and smaller gonads (Reyer et
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al. 1986; Schmidt et al. 1991; Schoech et al. 1991; Poiani and Fletcher 1994). However, the extent to which lower testosterone levels in helpers are merely correlated with their younger age, sexual immaturity or lack of breeding opportunities rather than being due to their subordinate status per se remains unclear (but see Reyer et al. 1986). To date, most studies of the role of testosterone in avian life histories have used model systems that exhibit either monogamy with high paternal care or polygyny with low paternal care, in either cooperative or pairbreeding systems. In contrast, superb fairy-wrens, Malurus cyaneus, provide the opportunity to examine the testosterone profile in a species that exhibits features that are conventionally associated with divergent hormone patterns. Superb fairy-wrens are cooperative breeders that combine a monogamous social system and high paternal care with a mating system based on extra-group fertilisation and intense sexual selection. Fairy-wrens live as pairs that can be assisted in raising their offspring by up to four behaviourally subordinate but reproductively competent male helpers. Males are seasonally dichromatic and all males develop a colourful nuptial plumage in their first breeding season (Mulder et al. 1994). Most (76%) offspring in the population are sired by males from outside their social group and the successful extragroup sires are those that develop their colourful plumage long before the start of breeding (Dunn and Cockburn 1999). While in nuptial plumage, all males frequently visit extra-group females to perform courtship displays (Mulder 1997). Despite consistently high levels of extra-group courtship and mating, males contribute substantially to offspring care, providing up to 50% of nestling feeds (Green et al. 1995; Dunn and Cockburn 1996). We studied the annual pattern of testosterone levels in relation to the social and mating system in superb fairywrens. We aimed to establish whether this pattern correlates with either the behavioural or the genetic mating system, that is, whether the testosterone profile resembles that of a monogamous, biparental species or a polygynous species with continued high investment in mating. We determined seasonal testosterone levels in males with and without paternal duties while within- and extragroup mating opportunities varied. In addition, we aimed to establish if male testosterone levels were correlated with their attractiveness to extra-group females, measured as the date of the moult into colourful nuptial plumage. Finally, we investigated the relationship between male social status and testosterone level by comparing similar-aged males living in pairs as dominant or subordinate males in groups. Our findings suggest that the testosterone profile is largely determined by the acquisition of the nuptial plumage and the mating system, while subtle differences in testosterone levels are related to 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 allpurpose territories as socially monogamous pairs which in 60% of cases are assisted by one to four helper males. In groups, the senior male is behaviourally dominant over the younger, subordinate helpers, and dominance is usually achieved by orderly queuing: when the dominant male dies, the next-oldest helper male automatically becomes dominant. Regardless of social status, all males are capable of breeding at 1 year of age when they develop a fullsized cloacal protuberance (see below; Mulder and Cockburn 1993), the ability to fertilise eggs (Dunn and Cockburn 1999) and a colourful nuptial plumage (Mulder and Magrath 1994). Male plumage alternates between dull brown eclipse plumage and iridescent blue-and-black nuptial plumage. Whereas males moult synchronously into eclipse plumage in late summer, they initiate moult into nuptial plumage at any time from early autumn until late spring (March until November; Dunn and Cockburn 1999). By the end of spring, all males have developed a complete nuptial plumage, and there is little variability in the extent or intensity of the plumage attained (A. Cockburn, unpublished data). In spring, all males also develop a cloacal protuberance, a large bulbous swelling that contains the seminal glomera, the site of sperm storage (Mulder and Cockburn 1993). Early acquisition of the nuptial plumage is an age- and condition-dependent indicator of male quality (Dunn and Cockburn 1999; Peters 2000): males moult progressively earlier until they are 5 years old, whereas they delay moult in harsh winters (Mulder and Magrath 1994). Testosterone levels rise during the moult, and males in blue plumage always have elevated testosterone, even in mid-winter, despite an immune suppressive effect of testosterone (Peters 2000; Peters et al. 2000). Individual males show high consistency in relative moult date between years (Mulder and Magrath 1994). Male reproductive success is heavily skewed: males that complete the nuptial plumage at least 1 month prior to the start of the breeding season are strongly preferred as extra-group mates (Dunn and Cockburn 1999). Despite year-round territoriality and social stability, extragroup matings are extraordinarily common (93% of nests, 76% of offspring; Mulder et al. 1994). Females are in control of extragroup mating (Double and Cockburn 2000) and males court females intensely: males of all ages and social classes engage in frequent (average 15–20 per day; Green et al. 1995) courtship displays directed exclusively at extra-group females (Mulder 1997). When nestlings require care, dominant males can maintain a higher rate of courtship displays because helpers compensate for loss of nestling care (Green et al. 1995). However, this comes at a cost: helpers also liberate females from constraints on extra-group mate choice, and dominant males in groups lose more paternity to extragroup sires than do males in unassisted pairs (Mulder et al. 1994). Study population All males for this study were taken from a colour-banded population in and around the Australian National Botanic Gardens (ANBG) Canberra, Australia. The ANBG is a reserve of dry sclerophyll forest and irrigated plantations of Australian native flora. Superb fairy-wrens have been studied at the ANBG since 1988 and most birds are of known age. Between 1996 and 1998, approximately 90 breeding groups were monitored each year. Group composition and dominance status of resident males were surveyed year-round for all social groups. During the breeding season, initiation and progress of the breeding activities of all females were followed closely in daily censuses. During the non-breeding season, all males were monitored for signs of initiation, progress and completion of the pre-nuptial moult (for a detailed description see Dunn and Cockburn 1999). Although females may initiate up to nine clutches per season, nest predation is high such that they
521 Table 1 Testosterone levels, mating opportunities and male plumage status during the three phases of the superb fairy-wren breeding cycle Phase
Number of males captured
Testosterone (ng/ml)
Number of fertile femalesa
Plumage
Mean (SD, maximum)
Mean (SD, maximum)
Brown
Moult
Blue
Pre-breeding (1 June–31 September)
125
0.30 (0.35, 2.4)
1 (2, 9)b
57
20
48
Breeding (1 October–14 January)
68
0.45 (0.63, 3.3)
14 (6, 29)
0
1
67
Post-breeding (15 January–31 May)
36
0.11 (0.05, 0.3)
0 (0, 0)
23
12
1
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 penultimate egg)
b A small number of females initiated breeding exceptionally early in 1998; in 1996 and in 1997, no female became fertile before 29 October
fledge at most three broods per year (A. Cockburn, unpublished data). Plasma sampling Birds were captured in mist nets between 0550–1130 hours (median 0730 hours) and weighed to the nearest 0.1 g using a 30-g Pesola spring balance. We measured head-bill and tarsus length as well as the length, width and depth of the cloacal swelling (Mulder and Cockburn 1993) to the nearest 0.05 mm using Vernier calipers. We estimated the percentage of visible coloured nuptial feathers (Dunn and Cockburn 1999). A small (60–140 µl) blood sample was collected from the brachial vein into heparinised capillary tubes. Tubes were stored vertically on ice until they were transported to the laboratory (within 5 h) and spun in a microhaematocrit centrifuge for 3 min at 12,000 rpm. Haematocrit was measured and the plasma separated from the packed cells and stored at –70 C. Blood samples were taken as soon as possible after capture (median=20 min, mean=25 min, SE=0.8, range 5–85 min). Because testosterone levels can vary during the day and can be affected by long delays between capture and blood sampling (Wingfield et al. 1982), we initially included the time of capture and the delay to sampling in all statistical analyses. There was no evidence for an effect of time of day or of a linear or nonlinear effect of the delay between capture and sampling on testosterone levels (all P>0.25). Testosterone analysis Plasma testosterone concentrations were determined within 10 months of plasma collection using a commercial radioimmunoassay (Pantex, Santa Monica, Calif.). This assay shows high specificity for testosterone and 7.9–10.5% intra-assay and 8.1–12% inter-assay variability. We modified this assay to accommodate small plasma volumes and low testosterone titres by halving the volumes of all other assay reagents, thus effectively doubling the recommended sample volume and increasing assay sensitivity. Plasma samples were analysed in two replicates of 10–30 µl each and when replicate values differed by more than 10%, the samples were excluded from analysis. The effective range of the assay was conservatively truncated below 0.10 ng/ml (approximately 90% binding) and testosterone values below the assay detection limit were set at 0.10 ng/ml accordingly (Peters et al. 2000). Statistical analysis The superb fairy-wren breeding cycle consists of three phases that are distinct in terms of male moult, female breeding activity and
Fig. 1 Annual variation in testosterone levels in male superb fairy-wrens (a) and the number of males captured in blue plumage and number of fertile females in the population (b). Males were captured between February 1996 and September 1998. Testosterone levels and proportion of blue males are monthly means±SE of all males sampled during the study; numbers refer to sample sizes. Number of fertile females are monthly means±SE of the daily total number of females (out of 85–90) in the population that were between 6 days before the start of laying and the day of the penultimate egg 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-breeding phase showed negligible variation. Since the temporal testosterone pattern differed substantially during the pre-breeding and breeding phase (Fig. 1), we developed separate multiple-regression models for these phases to determine which variables best explained the testosterone profile (see below for details).
522 For all analyses, testosterone concentrations were transformed by natural logarithm to fit standard least-squares models. We used a top-down approach in all multiple regression models, initially including all explanatory variables (see below for details) and progressively eliminating non-significant terms (P>0.05). All eliminated variables were reintroduced to the final model to confirm the lack of contribution, and statistical details presented (F, dfs, P-values) are based on the change in variance resulting from addition of the term to the final model. Residual plots and normal probability plots were examined for unequal variance and deviations from normality among residuals to verify the validity of the final model. All results presented (means±SE; back-transformed where appropriate) are predicted by a model containing all significant 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 of these were sampled in different years (mean±SE number of days between captures: 327±70). In addition, a separate analysis of 13 males that had been sampled twice within 6 weeks (from a total of 167 males sampled across the three periods) revealed no evidence for a correlation between repeated samples (A. Peters, unpublished data). In view of the lack of evidence for dependence and the small number and the long time span between repeated samples, we treated samples as independent. Before examining the significance of any parameters, we determined which function of sampling date and year best summarised the temporal pattern of testosterone. The model was derived through an iterative process by first fitting a curve to the relationship between testosterone and date for each year separately. Initially, we fitted separate fourth-order smoothing functions to the date, one for each year. We then progressively reduced the complexity of the functions until further reduction resulted in a significant (P
