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Testosterone and corticosterone during the breeding cycle of equatorial and European stonechats (Saxicola torquata axillaris and S. t. rubicola)
Hormones and Behavior 50 (2006) 779 – 785 www.elsevier.com/locate/yhbeh
Testosterone and corticosterone during the breeding cycle of equatorial and European stonechats (Saxicola torquata axillaris and S. t. rubicola) Wolfgang Goymann a,⁎, Dirk Geue a , Ingrid Schwabl a , Heiner Flinks b , Dieter Schmidl a , Hubert Schwabl c , Eberhard Gwinner a,† a
Max Planck Institute for Ornithology, Department of Biological Rhythms and Behaviour, Von-Der-Tann-Str. 7, D-82346 Andechs, Germany b Am Kuhm 19, D-46325 Borken, Germany c Center for Reproductive Biology, School of Biological Sciences, Washington State University, Pullman, WA 99164, USA Received 23 May 2006; revised 4 July 2006; accepted 6 July 2006 Available online 28 August 2006
Abstract Northern-temperate male birds show seasonal changes in testosterone concentrations with a peak during the breeding season. Many tropical birds express much lower concentrations of testosterone with slight elevations during breeding. Here we describe testosterone and corticosterone concentrations of male stonechats from equatorial Kenya during different substages of breeding and molt. This tropical species has a short breeding season of approximately 3 months. We compare their hormone concentrations to previously published data of males of a northerntemperate relative, the European stonechat, also a seasonal breeder but with a breeding season of approximately 5 months. Equatorial stonechats show a pronounced peak of testosterone during the nest-building and laying stage. During all other stages, testosterone concentrations are low, similar to other year-round territorial tropical bird species. Corticosterone concentrations peak also during the nest-building and laying stage suggesting that this period of maximum female fecundity is a demanding period for the male. Equatorial stonechats have significantly lower concentrations of testosterone than European stonechats during all stages, except during the nest-building and laying stage. During this stage of maximum female fertility, testosterone levels tend to be higher in equatorial than in European stonechats. Our results suggest that equatorial stonechats belong to a group of tropical bird species that are characterized by a short breeding season and a brief high peak of testosterone during the female's fertile period. Such brief, but substantial peaks of testosterone may be common in tropical birds, but they may easily be missed if the exact breeding stage of individual birds is not known. © 2006 Elsevier Inc. All rights reserved. Keywords: Tropical birds; Androgens; Glucocorticoids; Seasonal breeding; Molt
Introduction Males of many northern-temperate bird species show seasonal changes in testosterone concentrations which are often paralleled by seasonal changes in aggressive and territorial behavior (Wingfield and Farner, 1993; Wingfield and Silverin, 2002). In contrast, many tropical birds studied so far express low concentrations of plasma testosterone, involving low amplitude cycles with possible slight elevations
⁎ Corresponding author. Fax: +49 8152 373 133. E-mail address: [email protected] (W. Goymann). † Deceased Sept. 7th 2004. 0018-506X/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2006.07.002
during times of breeding (Dittami, 1986; Dittami and Knauer, 1986; Dittami, 1987; Dittami and Gwinner, 1990; Levin and Wingfield, 1992; Hau et al., 2000; Lormée et al, 2000; Hau, 2001; Stutchbury and Morton, 2001; Wikelski et al., 2003; Goymann et al., 2004; Fedy and Stutchbury, 2006). Some tropical bird species, however, express seasonally high levels of testosterone comparable to northern latitude species (Moore et al., 2002, 2004; Goymann and Wingfield, 2004; Goymann et al., 2004). Dittami and Gwinner (1985) have described the annual pattern of testosterone secretion in the year-round territorial equatorial stonechat (Saxicola torquata axillaris) and found that testosterone was maintained at relatively high levels (above 1 ng/ml) throughout the year, despite the fact that gonads were regressed for at least four months of the year. A
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short-coming of that study was that only a few samples could be assigned to the exact breeding stage of the respective birds. Hence, the first aim of the current study was to re-examine seasonal patterns of testosterone of free-ranging equatorial stonechats during known life-history stages. We focussed on hormone patterns during specific stages of the life cycle, in particular pre-breeding, incubation, feeding-young and molt stages. We also examined patterns of circulating corticosterone to relate these to reproductive stage and testosterone levels in the two taxa. The second aim of this study was to compare the hormone patterns of equatorial stonechats with those of northerntemperate relatives. In their recent comparative study, Goymann et al. (2004) have demonstrated that the length of the breeding season is one of the major predictors of male maximum testosterone concentrations in tropical birds. Highly seasonally breeding tropical birds show elevations in testosterone similar in magnitude to their northern latitude congeners. Because of their extensive breeding range from northern Siberia (70°N) to the southern tip of Africa (30°S; (Helm et al., 2005), stonechats represent an ideal taxon to study differences between tropical and temperate birds. We therefore relate our findings on equatorial stonechats to previously published hormone patterns of northern-temperate European stonechats (Saxicola torquata rubicola; Schwabl et al., 2005). Although the equatorial and the European stonechat are treated as subspecies and readily interbreed in captivity (Helm et al., 2005), molecular studies suggest that they might be considered different though closely related species (Wink et al., 2002a,b). Equatorial stonechats in Kenya are single-brooded, although they can raise multiple broods in captivity ((König and Gwinner, 1995). While the breeding season of a population covers approximately 5 months, an individual pair raises only one clutch of typically 3 young within a period of less than 3 months (Dittami and Gwinner, 1985; Helm et al., 2005). In contrast, European stonechats are multiple-brooded and raise up to three clutches of typically 5 young per clutch within a period of 5–6 months (Helm et al., 2005; Schwabl et al., 2005). Because of their short breeding season, we predicted that equatorial stonechats would show peak concentrations of testosterone similar to those of their European relatives. A recent study on captive and individually housed stonechats has shown, however, that peak concentrations of testosterone metabolites measured from droppings were significantly lower in equatorial than temperate stonechats (Roedl et al., 2004), suggesting that equatorial stonechats may express lower peak levels of testosterone than stonechats from northern-temperate regions. We did not have any specific predictions for differences in corticosterone, as both species breed in relatively benign environments with opportunities for replacement clutches in case one breeding attempt should fail. However, differences in corticosterone secretion patterns may provide a basis for understanding potential differences in reproductive investment in different environments and may be useful for the interpretation of differences in testosterone profiles.
Materials and methods Study site Equatorial stonechats were studied between April and June 2002 and during February and March 2003 at the Mau escarpment (0°86′ S, 36° 0′ E; ∼2500 m above sea level) and south of Olkalau (0°14′ S, 36°30′ E; ∼ 2300 m above sea level) an area between the Mau escarpment and the Aberdare mountains, Kenya. A total of 86 birds were caught during the following breeding life-history substages: pre-breeding, nest-building and egg-laying, incubation, feeding-young and during molt using mealworm-baited spring traps (N = 23) or spring traps with a mounted decoy (N = 63). This sample includes five individuals that were caught during 2 different stages, but were included as independent sample points in the analyses. Excluding the second samples of these five individuals did not change the results, hence we decided to keep all data in the analyses. Twenty-five additional birds were caught during an unknown breeding stage with mealworm-baited spring traps (N = 6) or spring traps with a mounted decoy (N = 19). These birds were not included in the hormonal analyses. The time (mean ± 95% confidence interval) between placing the decoy and catching the bird was 12.7 ± 2.7 min (N = 82, range 1– 70 min). The time period between males attacking the decoy and capture was 2.3 ± 0.9 min (N = 69, range 0–17 min). We compared hormone concentrations of equatorial stonechats with those reported previously by Schwabl et al. (2005) for European stonechats breeding in Northwestern Germany (51°47′ and 51°48′ N and 6°01′ and 7°08′ E). To standardize the comparison of single-brooded equatorial with multiple-brooded European stonechats, we only used data from the first broods of European stonechats. Since for the European population corticosterone data are available only for males during nestbuilding and laying and feeding-young, the comparison of corticosterone concentrations is limited to these two substages.
Blood sampling Blood samples of focal males were obtained immediately after capture (mean ± 95% CI: 6.3 ± 0.6 min) from the left wing vein using a sterile 23-gauge needle. Blood (150–200 μl) was collected into heparinized micropipettes, immediately centrifuged at 9000 rpm for 10 min and the plasma stored on dryice in the field until return to the field station at the end of the day. Then, the plasma was stored in liquid nitrogen, transported on dry-ice to Germany and kept at −70°C until analysis. Capture of stonechats and blood sampling procedures were authorized by Kenyan authorities and were conducted in accordance with the regulations regarding care and use of laboratory and wild animals in Kenya and Germany.
Steroid hormone measurement Testosterone (T) and corticosterone of equatorial stonechats were measured by radioimmunoassay (RIA) after partial purification on diatomaceous earth/glycol columns, using a modification (Goymann et al., 2001; Goymann et al., 2006) of the method described by Wingfield and Farner (1975). Data from European stonechats were obtained with the same method and were measured in the same laboratory, but testosterone concentrations were measured with a different antibody a long time ago (Wien Laboratories, Succasunna, NJ; Schwabl et al., 2005). Unpublished validation experiments have shown that, when hormones are separated with column cromatography, the two antibodies measure the exact same amount of testosterone (M. Wikelski and M. Hau, unpublished data), suggesting that it is possible to directly compare the data. Testosterone and corticosterone antisera were obtained from Esoterix Endocrinology (Calabasas Hills, USA). Because we used chromatography to separate and purify steroids, cross-reactivity of these antisera with other steroids is negligible. Standard steroids were purchased from Sigma-Aldrich (Munich, Germany), and 3H-labeled steroids from Perkin Elmer (Rodgau, Germany). All chemicals used were of analytical grade. Aliquots of plasma from equatorial stonechats (40–100 μl) were brought up to 300 μl with double-distilled H2O and equilibrated for at least 4 h at 4°C with
W. Goymann et al. / Hormones and Behavior 50 (2006) 779–785 ∼ 1500 dpm of 3H-testosterone (Perkin Elmer NET-553) and 3H-corticosterone (Perkin Elmer NET-399). Then, 4 ml dichloromethane was added to the samples. After overnight equilibration, the organic phase was separated from the aqueous phase by plunging the extraction tubes into a methanol-dry ice bath and decanting the dichloromethane phase. After a second extraction with 2 ml of dichloromethane, the samples were dried under a stream of nitrogen at 39°C. The extracts were resuspended in 2 × 250 μl 2% freshly redistilled ethylacetate in isooctane and transferred to the top of chromatography columns containing a mixture of diatomaceous earth:propylene glycol:ethylene glycol (6:1.5:1.5, w:v:v). Steroid hormones were separated on the basis of their polarity by eluting columns with 4 ml portions of increasing concentrations of ethyl acetate (2%, 10%, 20%, 40% and 50% EtAc) in isooctane. Testosterone was eluted in the third (20% EtAc) and corticosterone in the fifth fraction (50% EtAc). Collected fractions were dried at 39°C and then redissolved in 300 μl phosphate-buffered saline with 1% gelatine (PBSG) and left overnight at 4°C to equilibrate. Aliquots (80 μl) of each fraction and sample were transferred to scintillation vials, mixed with 4 ml scintillation fluid (Packard Ultima Gold) and counted to an accuracy of 2–3% in a Beckman LS 6000 βcounter to estimate individual extraction recoveries. The remainder was stored at − 40°C until RIA was conducted. Standard curves were set up by serial dilution of stock standard solutions with a concentration range of 0.4–200 pg for testosterone and 2–1000 pg for corticosterone in duplicates. Dilutions of the respective antiserum (1/200 for testosterone No. T3-125, 1/125 for corticosterone No. B3-163, both from Esoterix Endocrinology, Calabasas Hill, CA), were added to the standard curve, controls and to duplicates of the respective sample fractions (2 × 100 μl). After 30 min, 13500 dpm of 3H-testosterone or 15000 dpm of 3H-corticosterone was added and samples incubated for 20 h at 4°C. Antibody-bound and free fractions of the respective hormone were separated at 4°C by adding 0.5 ml dextrancoated charcoal. After 14 min incubation with charcoal, samples were spun (3600×g, 10 min, 4°C) and supernatants decanted into scintillation vials at 4°C. After adding 4 ml scintillation liquid (Packard Ultima Gold), vials were counted. Standard curves and sample concentrations were calculated with Immunofit 3.0 (Beckman Inc. Fullerton, CA), using a four parameter logistic curve fit. The lower detection limit of the standard curves was determined as the first value outside the 95% confidence intervals for the zero standard (Bmax) and was 8 pg/ml (0.8 pg per tube) for testosterone and 100 pg/ml (10 pg/tube) for corticosterone. Non-detectable values (N = 19 for testosterone and N = 1 for corticosterone) were assumed to be equivalent to the lower detection limits, thus giving a conservative estimate of hormone levels. The intra-assay variations were 3.1% and 3.9% for testosterone, and 6.2% and 14.7% for corticosterone. The inter-assay variation was 1.1% for testosterone and 1.8% for corticosterone. Individual recoveries for each hormone after column separation were calculated as percent activity eluted from the columns of total activity added prior to extraction with dichloromethane. Mean (±95% CI) recoveries were 55.6 ± 2.7% for testosterone and 90.1 ± 0.5% for corticosterone (N = 129).
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Results Equatorial stonechats Testosterone concentrations of equatorial male stonechats significantly varied with breeding stage (F4,79 = 8.33, p < 0.001) and were significantly higher during nest-building and laying than during all other stages (post hoc tests p < 0.02 for all comparisons, see Fig. 1a). Testosterone was lowest during molt (post hoc tests: incubation-molt: p = 0.02, pre-breeding-molt: p = 0.07; Fig. 1a). Testosterone concentrations did not significantly differ between equatorial stonechats caught with decoys and those caught with mealworm-baited traps (F1,79 = 2.94, p = 0.09; Fig. 1a). Time between capture and blood-sampling did not affect testosterone concentrations in an analysis of both trapping methods combined (F1,79 = 0.54, p = 0.46). Overall, the statistical model explained 34% of the variance in the data. Considering only birds caught with a decoy, breeding stage still had a significant effect on testosterone levels (F4,55 = 4.163, p = 0.005), but in this subset the time period between placing the decoy and capture of the bird had a significant effect on testosterone concentrations (F1,55 = 5.099, p = 0.028; Fig. 2a),
Statistics Statistical analyses were performed with Systat 11 (Systat Software Inc.). Models were tested for normality and equality of variances. If possible, data were transformed to meet criteria for parametric statistical tests and analyzed with general linear models (GLM). Initially, interaction terms were included in the models, and subsequently removed when they did not show any effect (p > 0.1; Grafen and Hails, 2002). Post hoc tests were done using the Bonferroni method implemented in the GLM of Systat. All models were tested for normal distribution of the residuals and equality of variances. To meet this criteria, testosterone concentrations were log10-transformed. Corticosterone concentrations were highly skewed and transformed using a Box–Cox transformation (formula: x′ = (x0.408 − 1) / 0.408 for the analysis of corticosterone data of equatorial stonechats and x′ = (x0.174 − 1) / 0.174 for the comparison between equatorial and European stonechats). Data are presented as mean ± 95% CI, the significance level was set at α = 0.05 and all p values are reported for twotailed tests. Transformed data are graphically represented as back-transformed means ± 95% CI, resulting in asymmetric representation of confidence intervals. Following recommendations of Wilkinson and Task Force on Statistical Inference (1999), we provided estimates of effect sizes (r2) along with each statistical test.
Fig. 1. Mean (± 95% CI) testosterone (a) and corticosterone (b) concentrations of equatorial stonechats during different stages of the breeding cycle caught with a mounted decoy (white bars) or with food-baited traps (grey bars). Numbers above bars indicate sample sizes.
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If we considered only birds caught with a decoy, breeding stage did not have a significant impact on corticosterone (F4,55 = 1.869, p = 0.129), but the time period between placing the decoy and capture of the bird was still significant (F1,55 = 6.192, p = 0.016; Fig. 2b) with birds caught sooner expressing lower levels of corticosterone than birds caught later. Considering only birds for which we had information about the time period between attacking the decoy and capture, breeding stage (F4,44 = 1.552, p = 0.204) and time between attacking the decoy and capture did not have a significant impact (F1,44 = 0.382, p = 0.539) on corticosterone levels, but there still was a trend of the time period between placing the decoy and capture (F1,44 = 3.352, p = 0.074; r2 = 0.18). Overall, testosterone and corticosterone concentrations did not correlate (Pearsons r = 0.13, p = 0.25, N = 86). Comparison between equatorial and European stonechats
Fig. 2. Testosterone (a) and corticosterone (b) concentrations of male equatorial stonechats in relation to the time elapsed between placing the decoy and capture.
with birds caught sooner expressing higher levels of testosterone than birds caught later. This statistical model explained 31% of the variance in the data. Considering only birds for which we had information about the time period between attacking the decoy and capture, breeding stage (F4,44 = 4.505, p = 0.004) and time between placing the decoy and capture had a significant effect (F1,44=5.404, p=0.025), but there was no relationship of the time period between attacking the decoy and capture with testosterone concentrations (F1,44 = 2.737, p = 0.110). This model explained 36% of the variance. Corticosterone concentrations of equatorial stonechats significantly varied with breeding stage (F4,75 = 3.87, p = 0.007) if we consider all birds. Corticosterone concentrations during nest-building and laying were significantly higher than during pre-breeding (post hoc test: p = 0.008, Fig. 1b) and they tended to be higher than during incubation (post hoc test: p = 0.07). Corticosterone concentrations were not affected by capture method (F1,75 = 0.98, p = 0.32), but by the period of time between capture and blood-sampling (F1,75 = 9.52, p = 0.003). The interaction between breeding stage and the capture method was also significant (F4,75 = 3.93, p = 0.006). This was mainly due to much lower levels of corticosterone in mealworm-trap caught pre-breeding males than in males caught during the same stage using a decoy (Fig. 1b). This model explained 40% of the variance in the data.
Overall, equatorial stonechats showed significantly lower testosterone concentrations than Europeans (F1,132 = 4.160, p = 0.043; Fig. 3). Testosterone varied significantly with breeding stage (F4,132 = 19.551, p < 0.0001) with testosterone being significantly higher during nest-building and laying than during all other stages (post hoc tests, p < 0.05 for all comparisons; Fig. 3). The interaction between taxon and breeding stage was not significant (F4,132 = 2.129, p = 0.08; Fig. 3). But equatorial stonechats had lower levels of testosterone than European stonechats during all breeding stages except for the nest-building and laying stage, when their levels were higher. This statistical model explained 44% of the overall variance in the data. Data for a comparison of corticosterone levels were only available for the incubation and feeding-young substages. Equatorial male stonechats had significantly higher corticosterone concentrations (F1,54 = 7.516, p = 0.008) than European males regardless of whether their females were incubating
Fig. 3. Comparison of mean (±95% CI) testosterone concentrations in equatorial (grey bars) and European stonechats (white bars). Numbers above bars indicate sample sizes.
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(Mean [− 95% CI; + 95% CI]: Equatorials: 7.8 ng/ml [6.0; 10.0], Europeans: 6.0 ng/ml [3.0; 11.2]) or whether the pair was feeding-young (Equatorials: 10.4 ng/ml [8.3; 13.0], Europeans 5.6 ng/ml [3.2; 9.3]). Breeding stage did not have a significant influence (F1,54 = 2.005, p = 0.163; r 2 = 0.15). Discussion In this study, we showed that stonechats from equatorial Kenya express high levels of testosterone during the nestbuilding and laying stage and low levels during all other breeding stages and postnuptial molt. Consistent with the previous study by Dittami and Gwinner (1985), we found the highest levels of testosterone during nest-building and laying. With an average of 2.8 ng/ml, the levels of the current study were approximately 1 ng/ml higher than the levels reported by Dittami and Gwinner (1985). During all other breeding stages and during postnuptial molt, the testosterone levels of the current study were much lower than those reported by Dittami and Gwinner (1985). They reported that the concentrations of testosterone remained close to or above 1 ng/ml year-round, but the breeding stage of most birds caught was not known. Our study suggests that equatorial stonechats have a single short and high peak of testosterone during nest-building and laying, levels of far less than 1 ng/ml during all other substages of the breeding life-history stage, and undetectable levels during molt. We do not know why the pattern of testosterone levels differs between the Dittami and Gwinner (1985) and our current study. Population differences are unlikely, as the two study sites are only few kilometers apart. One explanation might be that Dittami and Gwinner (1985) conducted their study during two exceptionally dry years (J. Dittami, personal communication), perhaps explaining the lower levels during the nest-building and laying stage and the higher levels in other stages. The pattern we found in equatorial stonechats was in part similar to that of most other tropical birds with year-round territoriality: they express low levels of testosterone during most of the year (Levin and Wingfield, 1992; Hau et al., 2000; Wikelski et al., 2003; Day et al., 2006; Fedy and Stutchbury, 2006). In contrast to these other tropical birds, however, equatorial stonechats showed a pronounced and high peak of testosterone during the period of nest-building, i.e. when females are receptive and eggs are fertilized. This peak in testosterone may either be triggered by increased male–male competition or by courtship including mate-guarding and female sexual behavior (Harding, 1981; Moore, 1982, 1983; Wingfield and Farner, 1993; Wingfield and Silverin, 2002; Wiley and Goldizen, 2003). Possibly, nest-building was the most demanding stage for male stonechats, because their corticosterone concentrations peaked during this breeding stage as well. Although the corticosterone concentrations we measured cannot be considered baseline (Romero and Reed, 2005), the levels we found were similar to the baseline levels collected during an earlier field study of equatorial stonechats in Tanzania (Scheuerlein et al., 2001). In this earlier study, males that had to share their territory with a fiscal shrike (Lanius collaris), a predator of small passerine birds and their nestlings,
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had higher corticosterone concentrations than males in territories without shrikes (Scheuerlein et al., 2001). The corticosterone concentrations of Kenyan males in the nestbuilding and laying stage were similar to those of Tanzanian males cohabiting with a shrike. This similarity renders further support for the idea that the nest-building and laying stage, during which the eggs were fertilized, was a demanding stage for males. Our birds were either caught with food-baited traps or by placing a mounted decoy into the territory. Overall, the pattern of testosterone and corticosterone secretion did not differ between capture methods, suggesting that the major determinant of testosterone and corticosterone secretion was the lifehistory stage and not the capture method. Interestingly, within the group that was caught with a decoy, testosterone concentrations decreased as the exposure time to the decoy increased. In contrast, corticosterone concentrations increased with exposure time. What does that mean? There are two possible explanations: First, testosterone may decrease and corticosterone may increase during a territorial challenge. Similar patterns have been found in northern pintails (Anas acuta), great tits (Parus major) and blue tits (Cyanistes caeruleus; Sorenson et al., 1997; Van Duyse et al., 2004; M. Landys, W. Goymann, M. Raess and T Slagsvold, unpublished data). Second, individuals with high testosterone and low corticosterone levels may be more likely to detect a decoy and react faster to it than individuals expressing low testosterone and high corticosterone levels. In equatorial stonechats, the latter explanation is more likely, as we did not find a relationship between testosterone or corticosterone and the time between attacking the decoy and capture. However, most birds were caught within less than 3 min after they had attacked the mounted decoy and this time may not have been long enough to mount a hormonal response (Wingfield and Wada, 1989). Tropical and temperate comparison Testosterone concentrations of free-ranging equatorial stonechats were significantly lower than those of northern-temperate stonechats during all breeding stages, except during the nestbuilding and laying stage. In fact, concentrations of testosterone during the nest-building and laying stage tended to be higher in equatorial stonechats than in their northern-temperate relatives. If we had included data for second and third broods of European stonechats, this difference during nest-building and laying would have been significant, because testosterone concentrations of European stonechats during nest-building and laying are lower during second and third broods compared to the first brood of the year. These results are in line with the idea that tropical birds generally have lower levels of testosterone than northern-temperate species (see references in the introduction and review in Stutchbury and Morton, 2001). Equatorial stonechats are territorial year-round and hence, testosterone may play only a minor role for territory establishment and maintenance. Our results also support the hypothesis that highly seasonal breeding is associated with high peak levels of
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testosterone at both low and high latitudes in stonechats. This is in accordance with the comparative study of Goymann et al. (2004) who found that the length of the breeding season is the major determinant of peak testosterone concentrations in tropical birds: the shorter the breeding season, the higher the peak levels of testosterone. Pairs of equatorial stonechats raise only one clutch per breeding season, which takes them less than 3 months. Hence, their breeding season is shorter than that of European stonechats which raise 2–3 clutches in a period of roughly 5 months. The results of this study suggest that equatorial stonechats belong to the group of tropical species, that combine a short breeding season with high peak levels of testosterone (Goymann et al., 2004). Because peak levels of testosterone coincide with the period of maximum fertility of the female, the pronounced increase in testosterone may either be stimulated by receptive behavior of the female or by increased risk of extra-pair behavior during this critical period. Within a taxon, similar results have been found by Moore et al. (2002) on birds of the genus Zonotrichia: during the breeding season equatorial rufous-collared sparrows (Zonotrichia capensis) mounted a higher testosterone response to GnRH injections than arctic Gambel's white-crowned sparrows (Z. leucophrys gambelii) and northern-temperate Puget Sound white-crowned sparrows (Z. leucophrys pugetensis). Possibly, there is also a phylogenetic aspect to the magnitude of testosterone levels in tropical birds. Testosterone patterns of tropical birds may differ between species with a phylogenetic origin in the temperate zones and species with a tropical phylogenetic history, i.e. tropical birds originating in temperate regions may express higher levels of testosterone than those with a true tropical phylogenetic history. Tentative evidence suggests that stonechats originated in the Eurasian tropics with an African branch diverging from this original line (Wink et al., 2002a,b; M. Wink, personal communication). Hence, stonechats are more likely of tropical than of temperate origin. For the rufous-collared sparrow, it seems to be unresolved whether it is of tropical or southern temperate origin (Zink and Blackwell, 1996, I. T. Moore, personal communication). Spotted antbirds (Hylophylax n. naevioides), another yearround territorial tropical bird species, are considered to have evolved in the tropics, but also they express brief periods with high levels of testosterone (Canoine et al., in press), suggesting that the phylogenetic history may be of only minor importance. We have previously measured testosterone metabolite concentrations from droppings of several stonechat populations in captivity (Roedl et al., 2004). Captive male equatorial stonechats kept individually in cages where they could hear but not see each other expressed a seasonal pattern in testosterone metabolite concentrations with higher levels during the breeding season and lower levels thereafter (Roedl et al., 2004). These captive equatorial stonechats generally had lower levels of testosterone metabolites than European stonechats kept under similar conditions. However, they did not show a pronounced peak in testosterone metabolite concentrations similar to the plasma testosterone peak that free-living conspecifics showed during the nest-building and laying stage. We may have missed this peak in the captive study
because we sampled birds in intervals of 4 weeks only. Alternatively, the peak may result from supplementary environmental cues (i.e. interactions with females or other males). Although tropical birds including equatorial stonechats respond to photoperiod (Gwinner and Dittami, 1985; Hau et al., 1998), it is still unclear which environmental cues drive seasonal timing of reproduction at or close to the equator where photoperiodic information is basically absent. Most likely equatorial stonechats are much more sensitive to supplementary information from the environment to time breeding and other life-history stages than their northerntemperate relatives (Gwinner and Scheuerlein, 1998; Scheuerlein and Gwinner, 2002) and hence it is conceivable that supplementary environmental cues or a female partner causes the seasonal testosterone peak. Corticosterone concentrations during the incubation and feeding stage were higher in African than in European male stonechats. Due to the limited sample size of European stonechats, these results should be considered preliminary. In conclusion, the pattern of testosterone in equatorial male stonechats follows the general pattern of many tropical birds with overall testosterone concentrations being lower than in northern-temperate birds. However, similar to other tropical birds with short breeding seasons, they express high levels of peak testosterone during the short period of maximal female fertility. Equatorial stonechats even had higher levels of testosterone during this critical period than their northerntemperate congeners. The results of this study underline the importance of carefully sampling substages of the breeding lifehistory stage. Tropical birds may express high peak levels of testosterone, but due to the often lower breeding synchrony in the tropics (Stutchbury and Morton, 2001) such brief peaks may be easily missed if the exact breeding stage is not known. Acknowledgments This paper was written after the untimely death of Prof. Eberhard Gwinner. However, the project was planned and conducted by him with assistance of some of the coauthors. Therefore, Ebo Gwinner made a fundamental contribution to the writing of this paper, although he deceased before the data could be analyzed. We thank Nicole Geberzahn and Barbara Helm and two anonymous referees for reading and improving previous versions of the manuscript. Furthermore, we would like to thank Kenya Wildlife Service, the National Museum of Kenya and the Department for Ornithology for their cooperation. References Canoine, V., Fusani, L., Schlinger, B. A., Hau, M., in press. Low sex steroids, high steroid receptors: increasing the sensitivity of the nonreproductive brain. J. Neurobiol. 66. Day, L.B., McBroom, J.T., Schlinger, B.A., 2006. Testosterone increases display behaviors but does not stimulate growth of adult plumage in male goldencollared manakins (Manacus vitellinus). Horm. Behav. 49, 223–232. Dittami, J.P., 1986. Seasonal reproduction, molt and their endocrine correlates in two tropical Ploceidae species. J. Comp. Physiol., B 156, 641–647.
W. Goymann et al. / Hormones and Behavior 50 (2006) 779–785 Dittami, J.P., 1987. A comparison of breeding an molt cycles and life histories in two tropical starling species: the blue-eared glossy starling Lamprotornis chalybaeus and Rueppell's long-tailed glossy starling L. purpuropterus. Ibis 129, 69–85. Dittami, J.P., Gwinner, E., 1985. Annual cycles in the African stonechat Saxicola torquata axillaris and their relationship to environmental factors. J. Zool. 207, 357–370. Dittami, J.P., Knauer, B., 1986. Seasonal organization of breeding and molting in the Fiscal shrike Lanius collaris. J. Ornithol. 127, 79–84. Dittami, J., Gwinner, E., 1990. Endocrine correlates of seasonal reproduction and territorial behavior in some tropical passerines. In: Wada, M. (Ed.), Endocrinology of Birds: Molecular to Behavioral. Japan Scientific Society Press Springer, Tokyo and Berlin, pp. 225–233. Fedy, B.C, Stutchbury, B.J.M., 2006. Testosterone does not increase in response to conspecific challenges in the white-bellied antbird (Myrmeciza longipes), a resident tropical passerine. The Auk 123, 61–66. Goymann, W., Wingfield, J.C., 2004. Competing females and caring males. Sex steroids in African black coucals, Centropus grillii. Anim. Behav. 64, 733–740. Goymann, W., East, M.L., Hofer, H., 2001. Androgens and the role of female “hyperaggressiveness” in spotted hyenas (Crocuta crocuta). Horm. Behav. 39, 83–92. Goymann, W., Moore, I.T., Scheuerlein, A., Hirschenhauser, K., Grafen, A., Wingfield, J.C., 2004. Testosterone in tropical birds: effects of environmental and social factors. Am. Nat. 164, 327–334. Goymann, W., Trappschuh, M., Jensen, W., Schwabl, I., 2006. Low ambient temperature increases food intake and dropping production, leading to incorrect estimates of hormone metabolite concentrations in European stonechats. Horm. Behav. 49, 644–653. Grafen, A., Hails, R., 2002. Modern Statistics for the Life Sciences. Oxford Univ. Press, Oxford. Gwinner, E., Dittami, J., 1985. Photoperiodic responses in temperate zone and equatorial stonechats: a contribution to the problem of photoperiodism in tropical organisms. In: Follett, B.K., Ishii, S., Chandola, A. (Eds.), The Endocrine System and the Environment. Japan Scientific Society Press and Springer, Tokyo and Berlin, pp. 279–294. Gwinner, E., Scheuerlein, A., 1998. Seasonal changes in day-light intensity as a potential zeitgeber of circannual rhythms in equatorial stonechats. J. Ornithol. 139, 407–412. Harding, C.F., 1981. Social modulation of circulating hormone levels in the male. Am. Zool. 21, 223–232. Hau, M., 2001. Timing of breeding in variable environments: tropical birds as model systems. Horm. Behav. 40, 281–290. Hau, M., Wikelski, M., Wingfield, J.C., 1998. A neotropical forest bird can measure the slight changes in tropical photoperiod. Proc. R. Soc. B 265, 89–95. Hau, M., Wikelski, M., Soma, K.K., Wingfield, J.C., 2000. Testosterone and year-round territorial aggression in a tropical bird. Gen. Comp. Endocrinol. 117, 20–33. Helm, B., Gwinner, E., Trost, L., 2005. Flexible seasonal timing and migratory behavior: results from stonechat breeding programs. Ann. N. Y. Acad. Sci. 1046, 216–227. König, S., Gwinner, E., 1995. Frequency and timing of successive broods in captive African and European stonechats Saxicola torquata axillaris and S. t. rubicola. J. Avian Biol. 23, 247–254. Levin, R.N., Wingfield, J.C., 1992. The hormonal control of territorial aggression in tropical birds. Orn. Scand. 23, 284–291. Lormée, H., Jouventin, P., Lacroix, A., Lallemand, J., Chastel, O., 2000. Reproductive endocrinology of tropical seabirds: sex-specific patterns in LH, steroids, and prolactin secretion in relation to parental care. Gen. Comp. Endocrinol. 117, 413–426. Moore, M.C., 1982. Hormonal response of free-living male white-crowned sparrows to experimental manipulation of female sexual behavior. Horm. Behav. 16, 323–329.
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Moore, M.C., 1983. Effect of female sexual displays on the endocrine physiology and behaviour of male white-crowned sparrows, Zonotrichia leucophrys. J. Zool. 199, 137–148. Moore, I.T., Perfito, N., Wada, H., Sperry, T.S., Wingfield, J.C., 2002. Latitudinal variation in plasma testosterone levels in birds of the genus Zonotrichia. Gen. Comp. Endocrinol. 129, 13–19. Moore, I.T., Wada, H., Perfito, N., Busch, D.S., Hahn, T.P., Wingfield, J.C., 2004. Territoriality and testosterone in an equatorial population of rufous-collared sparrows, Zonotrichia capensis. Anim. Behav. 67, 411–420. Roedl, T., Goymann, W., Schwabl, I., Gwinner, E., 2004. Excremental androgen metabolite concentrations and gonad sizes in temperate zone vs. tropical Stonechats (Saxicola torquata spp.). Gen. Comp. Endocrinol. 139, 124–130. Romero, L.M., Reed, J.M., 2005. Collecting baseline corticosterone samples in the field: is under 3 min good enough? Comp. Biochem. Physiol., A 140, 73–79. Scheuerlein, A., Gwinner, E., 2002. Is food availability a circannual Zeitgeber in tropical birds? A field experiment on stonechats in tropical Africa. J. Biol. Rhythms 17, 171–180. Scheuerlein, A., Van't Hof, T.J., Gwinner, E., 2001. Predators as stressors? Physiological and reproductive consequences of predation risk in tropical stonechats (Saxicola torquata axillaris). Proc. R. Soc. B 268, 1575–1582. Schwabl, H., Flinks, H., Gwinner, E., 2005. Testosterone, reproductive stage, and territorial behavior of male and female European stonechats Saxicola torquata. Horm. Behav. 47, 503–512. Sorenson, L.G., Nolan, P.M., Brown, A.M., Derrickson, S.R., Monfort, S.L., 1997. Hormonal dynamics during mate choice in the northern pintail—A test of the challenge hypothesis. Anim. Behav. 54, 1117–1133. Stutchbury, B.J.M., Morton, E.S., 2001. Behavioral Ecology of Tropical birds. Academic Press, San Diego. Van Duyse, E., Pinxten, R., Darras, V.M., Arckens, L., Eens, M., 2004. Opposite changes in plasma testosterone and corticosterone levels following a simulated territorial challenge in male great tits. Behaviorology 141, 451–467. Wikelski, M., Hau, M., Robinson, W.D., Wingfield, J.C., 2003. Reproductive seasonality of seven neotropical passerine species. Condor 105, 683–695. Wiley, C.J., Goldizen, A.W., 2003. Testosterone is correlated with courtship but not aggression in the tropical buff-banded rail, Gallirallus philippensis. Horm. Behav. 43, 554–560. Wilkinson, L., Task Force on Statistical Inference, 1999. Statistical methods in psychology journals: guidelines and explanations. Am. Psychol. 54, 594–604. Wingfield, J.C., Farner, D.S., 1975. The determination of five steroids in avian plasma by radioimmunoassay and competitive protein-binding. Steroids 26, 311–327. Wingfield, J.C., Farner, D.S., 1993. Endocrinology of reproduction in wild species. In: Farner, D.S., King, J.R., Parkes, K.C. (Eds.), Avian Biology, vol. IX. Harcourt Brace and Company, London, pp. 163–327. Wingfield, J.C., Silverin, B., 2002. Ecophysiological studies of hormone– behavior relations in birds. In: Pfaff, D.W., Arnold, A.P., Etgen, A.M., Fahrbach, S.E., Rubin, R.T. (Eds.), Hormones, Brain and Behavior. Academic Press, San Diego, pp. 587–647. Wingfield, J.C., Wada, M., 1989. Changes in plasma levels of testosterone during male–male interactions in the song sparrow, Melospiza melodia: time course and specifity of response. J. Comp. Physiol., A 166, 189–194. Wink, M., Sauer-Gürth, H., Heidrich, P., Witt, H.-H., Gwinner, E., 2002a. A molecular phylogeny of stonechats and related turdids. In: Urquhart, E. (Ed.), Stonechats. Christopher Helm, London, pp. 22–30. Wink, M., Sauer-Gürth, H., Gwinner, E., 2002b. Evolutionary relationships of stonechats and related species inferred from mitochondrial-DNA sequences and genomic fingerprinting. Br. Birds 95, 349–355. Zink, R.M., Blackwell, R.C., 1996. Patterns of allozyme mitochondrial DNA, and morphometric variation in four sparrow genera. Auk 113, 59–67.
Testosterone and corticosterone during the breeding cycle of equatorial and European stonechats (Saxicola torquata axillaris and S. t. rubicola) Wolfgang Goymann a,⁎, Dirk Geue a , Ingrid Schwabl a , Heiner Flinks b , Dieter Schmidl a , Hubert Schwabl c , Eberhard Gwinner a,† a
Max Planck Institute for Ornithology, Department of Biological Rhythms and Behaviour, Von-Der-Tann-Str. 7, D-82346 Andechs, Germany b Am Kuhm 19, D-46325 Borken, Germany c Center for Reproductive Biology, School of Biological Sciences, Washington State University, Pullman, WA 99164, USA Received 23 May 2006; revised 4 July 2006; accepted 6 July 2006 Available online 28 August 2006
Abstract Northern-temperate male birds show seasonal changes in testosterone concentrations with a peak during the breeding season. Many tropical birds express much lower concentrations of testosterone with slight elevations during breeding. Here we describe testosterone and corticosterone concentrations of male stonechats from equatorial Kenya during different substages of breeding and molt. This tropical species has a short breeding season of approximately 3 months. We compare their hormone concentrations to previously published data of males of a northerntemperate relative, the European stonechat, also a seasonal breeder but with a breeding season of approximately 5 months. Equatorial stonechats show a pronounced peak of testosterone during the nest-building and laying stage. During all other stages, testosterone concentrations are low, similar to other year-round territorial tropical bird species. Corticosterone concentrations peak also during the nest-building and laying stage suggesting that this period of maximum female fecundity is a demanding period for the male. Equatorial stonechats have significantly lower concentrations of testosterone than European stonechats during all stages, except during the nest-building and laying stage. During this stage of maximum female fertility, testosterone levels tend to be higher in equatorial than in European stonechats. Our results suggest that equatorial stonechats belong to a group of tropical bird species that are characterized by a short breeding season and a brief high peak of testosterone during the female's fertile period. Such brief, but substantial peaks of testosterone may be common in tropical birds, but they may easily be missed if the exact breeding stage of individual birds is not known. © 2006 Elsevier Inc. All rights reserved. Keywords: Tropical birds; Androgens; Glucocorticoids; Seasonal breeding; Molt
Introduction Males of many northern-temperate bird species show seasonal changes in testosterone concentrations which are often paralleled by seasonal changes in aggressive and territorial behavior (Wingfield and Farner, 1993; Wingfield and Silverin, 2002). In contrast, many tropical birds studied so far express low concentrations of plasma testosterone, involving low amplitude cycles with possible slight elevations
⁎ Corresponding author. Fax: +49 8152 373 133. E-mail address: [email protected] (W. Goymann). † Deceased Sept. 7th 2004. 0018-506X/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2006.07.002
during times of breeding (Dittami, 1986; Dittami and Knauer, 1986; Dittami, 1987; Dittami and Gwinner, 1990; Levin and Wingfield, 1992; Hau et al., 2000; Lormée et al, 2000; Hau, 2001; Stutchbury and Morton, 2001; Wikelski et al., 2003; Goymann et al., 2004; Fedy and Stutchbury, 2006). Some tropical bird species, however, express seasonally high levels of testosterone comparable to northern latitude species (Moore et al., 2002, 2004; Goymann and Wingfield, 2004; Goymann et al., 2004). Dittami and Gwinner (1985) have described the annual pattern of testosterone secretion in the year-round territorial equatorial stonechat (Saxicola torquata axillaris) and found that testosterone was maintained at relatively high levels (above 1 ng/ml) throughout the year, despite the fact that gonads were regressed for at least four months of the year. A
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short-coming of that study was that only a few samples could be assigned to the exact breeding stage of the respective birds. Hence, the first aim of the current study was to re-examine seasonal patterns of testosterone of free-ranging equatorial stonechats during known life-history stages. We focussed on hormone patterns during specific stages of the life cycle, in particular pre-breeding, incubation, feeding-young and molt stages. We also examined patterns of circulating corticosterone to relate these to reproductive stage and testosterone levels in the two taxa. The second aim of this study was to compare the hormone patterns of equatorial stonechats with those of northerntemperate relatives. In their recent comparative study, Goymann et al. (2004) have demonstrated that the length of the breeding season is one of the major predictors of male maximum testosterone concentrations in tropical birds. Highly seasonally breeding tropical birds show elevations in testosterone similar in magnitude to their northern latitude congeners. Because of their extensive breeding range from northern Siberia (70°N) to the southern tip of Africa (30°S; (Helm et al., 2005), stonechats represent an ideal taxon to study differences between tropical and temperate birds. We therefore relate our findings on equatorial stonechats to previously published hormone patterns of northern-temperate European stonechats (Saxicola torquata rubicola; Schwabl et al., 2005). Although the equatorial and the European stonechat are treated as subspecies and readily interbreed in captivity (Helm et al., 2005), molecular studies suggest that they might be considered different though closely related species (Wink et al., 2002a,b). Equatorial stonechats in Kenya are single-brooded, although they can raise multiple broods in captivity ((König and Gwinner, 1995). While the breeding season of a population covers approximately 5 months, an individual pair raises only one clutch of typically 3 young within a period of less than 3 months (Dittami and Gwinner, 1985; Helm et al., 2005). In contrast, European stonechats are multiple-brooded and raise up to three clutches of typically 5 young per clutch within a period of 5–6 months (Helm et al., 2005; Schwabl et al., 2005). Because of their short breeding season, we predicted that equatorial stonechats would show peak concentrations of testosterone similar to those of their European relatives. A recent study on captive and individually housed stonechats has shown, however, that peak concentrations of testosterone metabolites measured from droppings were significantly lower in equatorial than temperate stonechats (Roedl et al., 2004), suggesting that equatorial stonechats may express lower peak levels of testosterone than stonechats from northern-temperate regions. We did not have any specific predictions for differences in corticosterone, as both species breed in relatively benign environments with opportunities for replacement clutches in case one breeding attempt should fail. However, differences in corticosterone secretion patterns may provide a basis for understanding potential differences in reproductive investment in different environments and may be useful for the interpretation of differences in testosterone profiles.
Materials and methods Study site Equatorial stonechats were studied between April and June 2002 and during February and March 2003 at the Mau escarpment (0°86′ S, 36° 0′ E; ∼2500 m above sea level) and south of Olkalau (0°14′ S, 36°30′ E; ∼ 2300 m above sea level) an area between the Mau escarpment and the Aberdare mountains, Kenya. A total of 86 birds were caught during the following breeding life-history substages: pre-breeding, nest-building and egg-laying, incubation, feeding-young and during molt using mealworm-baited spring traps (N = 23) or spring traps with a mounted decoy (N = 63). This sample includes five individuals that were caught during 2 different stages, but were included as independent sample points in the analyses. Excluding the second samples of these five individuals did not change the results, hence we decided to keep all data in the analyses. Twenty-five additional birds were caught during an unknown breeding stage with mealworm-baited spring traps (N = 6) or spring traps with a mounted decoy (N = 19). These birds were not included in the hormonal analyses. The time (mean ± 95% confidence interval) between placing the decoy and catching the bird was 12.7 ± 2.7 min (N = 82, range 1– 70 min). The time period between males attacking the decoy and capture was 2.3 ± 0.9 min (N = 69, range 0–17 min). We compared hormone concentrations of equatorial stonechats with those reported previously by Schwabl et al. (2005) for European stonechats breeding in Northwestern Germany (51°47′ and 51°48′ N and 6°01′ and 7°08′ E). To standardize the comparison of single-brooded equatorial with multiple-brooded European stonechats, we only used data from the first broods of European stonechats. Since for the European population corticosterone data are available only for males during nestbuilding and laying and feeding-young, the comparison of corticosterone concentrations is limited to these two substages.
Blood sampling Blood samples of focal males were obtained immediately after capture (mean ± 95% CI: 6.3 ± 0.6 min) from the left wing vein using a sterile 23-gauge needle. Blood (150–200 μl) was collected into heparinized micropipettes, immediately centrifuged at 9000 rpm for 10 min and the plasma stored on dryice in the field until return to the field station at the end of the day. Then, the plasma was stored in liquid nitrogen, transported on dry-ice to Germany and kept at −70°C until analysis. Capture of stonechats and blood sampling procedures were authorized by Kenyan authorities and were conducted in accordance with the regulations regarding care and use of laboratory and wild animals in Kenya and Germany.
Steroid hormone measurement Testosterone (T) and corticosterone of equatorial stonechats were measured by radioimmunoassay (RIA) after partial purification on diatomaceous earth/glycol columns, using a modification (Goymann et al., 2001; Goymann et al., 2006) of the method described by Wingfield and Farner (1975). Data from European stonechats were obtained with the same method and were measured in the same laboratory, but testosterone concentrations were measured with a different antibody a long time ago (Wien Laboratories, Succasunna, NJ; Schwabl et al., 2005). Unpublished validation experiments have shown that, when hormones are separated with column cromatography, the two antibodies measure the exact same amount of testosterone (M. Wikelski and M. Hau, unpublished data), suggesting that it is possible to directly compare the data. Testosterone and corticosterone antisera were obtained from Esoterix Endocrinology (Calabasas Hills, USA). Because we used chromatography to separate and purify steroids, cross-reactivity of these antisera with other steroids is negligible. Standard steroids were purchased from Sigma-Aldrich (Munich, Germany), and 3H-labeled steroids from Perkin Elmer (Rodgau, Germany). All chemicals used were of analytical grade. Aliquots of plasma from equatorial stonechats (40–100 μl) were brought up to 300 μl with double-distilled H2O and equilibrated for at least 4 h at 4°C with
W. Goymann et al. / Hormones and Behavior 50 (2006) 779–785 ∼ 1500 dpm of 3H-testosterone (Perkin Elmer NET-553) and 3H-corticosterone (Perkin Elmer NET-399). Then, 4 ml dichloromethane was added to the samples. After overnight equilibration, the organic phase was separated from the aqueous phase by plunging the extraction tubes into a methanol-dry ice bath and decanting the dichloromethane phase. After a second extraction with 2 ml of dichloromethane, the samples were dried under a stream of nitrogen at 39°C. The extracts were resuspended in 2 × 250 μl 2% freshly redistilled ethylacetate in isooctane and transferred to the top of chromatography columns containing a mixture of diatomaceous earth:propylene glycol:ethylene glycol (6:1.5:1.5, w:v:v). Steroid hormones were separated on the basis of their polarity by eluting columns with 4 ml portions of increasing concentrations of ethyl acetate (2%, 10%, 20%, 40% and 50% EtAc) in isooctane. Testosterone was eluted in the third (20% EtAc) and corticosterone in the fifth fraction (50% EtAc). Collected fractions were dried at 39°C and then redissolved in 300 μl phosphate-buffered saline with 1% gelatine (PBSG) and left overnight at 4°C to equilibrate. Aliquots (80 μl) of each fraction and sample were transferred to scintillation vials, mixed with 4 ml scintillation fluid (Packard Ultima Gold) and counted to an accuracy of 2–3% in a Beckman LS 6000 βcounter to estimate individual extraction recoveries. The remainder was stored at − 40°C until RIA was conducted. Standard curves were set up by serial dilution of stock standard solutions with a concentration range of 0.4–200 pg for testosterone and 2–1000 pg for corticosterone in duplicates. Dilutions of the respective antiserum (1/200 for testosterone No. T3-125, 1/125 for corticosterone No. B3-163, both from Esoterix Endocrinology, Calabasas Hill, CA), were added to the standard curve, controls and to duplicates of the respective sample fractions (2 × 100 μl). After 30 min, 13500 dpm of 3H-testosterone or 15000 dpm of 3H-corticosterone was added and samples incubated for 20 h at 4°C. Antibody-bound and free fractions of the respective hormone were separated at 4°C by adding 0.5 ml dextrancoated charcoal. After 14 min incubation with charcoal, samples were spun (3600×g, 10 min, 4°C) and supernatants decanted into scintillation vials at 4°C. After adding 4 ml scintillation liquid (Packard Ultima Gold), vials were counted. Standard curves and sample concentrations were calculated with Immunofit 3.0 (Beckman Inc. Fullerton, CA), using a four parameter logistic curve fit. The lower detection limit of the standard curves was determined as the first value outside the 95% confidence intervals for the zero standard (Bmax) and was 8 pg/ml (0.8 pg per tube) for testosterone and 100 pg/ml (10 pg/tube) for corticosterone. Non-detectable values (N = 19 for testosterone and N = 1 for corticosterone) were assumed to be equivalent to the lower detection limits, thus giving a conservative estimate of hormone levels. The intra-assay variations were 3.1% and 3.9% for testosterone, and 6.2% and 14.7% for corticosterone. The inter-assay variation was 1.1% for testosterone and 1.8% for corticosterone. Individual recoveries for each hormone after column separation were calculated as percent activity eluted from the columns of total activity added prior to extraction with dichloromethane. Mean (±95% CI) recoveries were 55.6 ± 2.7% for testosterone and 90.1 ± 0.5% for corticosterone (N = 129).
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Results Equatorial stonechats Testosterone concentrations of equatorial male stonechats significantly varied with breeding stage (F4,79 = 8.33, p < 0.001) and were significantly higher during nest-building and laying than during all other stages (post hoc tests p < 0.02 for all comparisons, see Fig. 1a). Testosterone was lowest during molt (post hoc tests: incubation-molt: p = 0.02, pre-breeding-molt: p = 0.07; Fig. 1a). Testosterone concentrations did not significantly differ between equatorial stonechats caught with decoys and those caught with mealworm-baited traps (F1,79 = 2.94, p = 0.09; Fig. 1a). Time between capture and blood-sampling did not affect testosterone concentrations in an analysis of both trapping methods combined (F1,79 = 0.54, p = 0.46). Overall, the statistical model explained 34% of the variance in the data. Considering only birds caught with a decoy, breeding stage still had a significant effect on testosterone levels (F4,55 = 4.163, p = 0.005), but in this subset the time period between placing the decoy and capture of the bird had a significant effect on testosterone concentrations (F1,55 = 5.099, p = 0.028; Fig. 2a),
Statistics Statistical analyses were performed with Systat 11 (Systat Software Inc.). Models were tested for normality and equality of variances. If possible, data were transformed to meet criteria for parametric statistical tests and analyzed with general linear models (GLM). Initially, interaction terms were included in the models, and subsequently removed when they did not show any effect (p > 0.1; Grafen and Hails, 2002). Post hoc tests were done using the Bonferroni method implemented in the GLM of Systat. All models were tested for normal distribution of the residuals and equality of variances. To meet this criteria, testosterone concentrations were log10-transformed. Corticosterone concentrations were highly skewed and transformed using a Box–Cox transformation (formula: x′ = (x0.408 − 1) / 0.408 for the analysis of corticosterone data of equatorial stonechats and x′ = (x0.174 − 1) / 0.174 for the comparison between equatorial and European stonechats). Data are presented as mean ± 95% CI, the significance level was set at α = 0.05 and all p values are reported for twotailed tests. Transformed data are graphically represented as back-transformed means ± 95% CI, resulting in asymmetric representation of confidence intervals. Following recommendations of Wilkinson and Task Force on Statistical Inference (1999), we provided estimates of effect sizes (r2) along with each statistical test.
Fig. 1. Mean (± 95% CI) testosterone (a) and corticosterone (b) concentrations of equatorial stonechats during different stages of the breeding cycle caught with a mounted decoy (white bars) or with food-baited traps (grey bars). Numbers above bars indicate sample sizes.
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If we considered only birds caught with a decoy, breeding stage did not have a significant impact on corticosterone (F4,55 = 1.869, p = 0.129), but the time period between placing the decoy and capture of the bird was still significant (F1,55 = 6.192, p = 0.016; Fig. 2b) with birds caught sooner expressing lower levels of corticosterone than birds caught later. Considering only birds for which we had information about the time period between attacking the decoy and capture, breeding stage (F4,44 = 1.552, p = 0.204) and time between attacking the decoy and capture did not have a significant impact (F1,44 = 0.382, p = 0.539) on corticosterone levels, but there still was a trend of the time period between placing the decoy and capture (F1,44 = 3.352, p = 0.074; r2 = 0.18). Overall, testosterone and corticosterone concentrations did not correlate (Pearsons r = 0.13, p = 0.25, N = 86). Comparison between equatorial and European stonechats
Fig. 2. Testosterone (a) and corticosterone (b) concentrations of male equatorial stonechats in relation to the time elapsed between placing the decoy and capture.
with birds caught sooner expressing higher levels of testosterone than birds caught later. This statistical model explained 31% of the variance in the data. Considering only birds for which we had information about the time period between attacking the decoy and capture, breeding stage (F4,44 = 4.505, p = 0.004) and time between placing the decoy and capture had a significant effect (F1,44=5.404, p=0.025), but there was no relationship of the time period between attacking the decoy and capture with testosterone concentrations (F1,44 = 2.737, p = 0.110). This model explained 36% of the variance. Corticosterone concentrations of equatorial stonechats significantly varied with breeding stage (F4,75 = 3.87, p = 0.007) if we consider all birds. Corticosterone concentrations during nest-building and laying were significantly higher than during pre-breeding (post hoc test: p = 0.008, Fig. 1b) and they tended to be higher than during incubation (post hoc test: p = 0.07). Corticosterone concentrations were not affected by capture method (F1,75 = 0.98, p = 0.32), but by the period of time between capture and blood-sampling (F1,75 = 9.52, p = 0.003). The interaction between breeding stage and the capture method was also significant (F4,75 = 3.93, p = 0.006). This was mainly due to much lower levels of corticosterone in mealworm-trap caught pre-breeding males than in males caught during the same stage using a decoy (Fig. 1b). This model explained 40% of the variance in the data.
Overall, equatorial stonechats showed significantly lower testosterone concentrations than Europeans (F1,132 = 4.160, p = 0.043; Fig. 3). Testosterone varied significantly with breeding stage (F4,132 = 19.551, p < 0.0001) with testosterone being significantly higher during nest-building and laying than during all other stages (post hoc tests, p < 0.05 for all comparisons; Fig. 3). The interaction between taxon and breeding stage was not significant (F4,132 = 2.129, p = 0.08; Fig. 3). But equatorial stonechats had lower levels of testosterone than European stonechats during all breeding stages except for the nest-building and laying stage, when their levels were higher. This statistical model explained 44% of the overall variance in the data. Data for a comparison of corticosterone levels were only available for the incubation and feeding-young substages. Equatorial male stonechats had significantly higher corticosterone concentrations (F1,54 = 7.516, p = 0.008) than European males regardless of whether their females were incubating
Fig. 3. Comparison of mean (±95% CI) testosterone concentrations in equatorial (grey bars) and European stonechats (white bars). Numbers above bars indicate sample sizes.
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(Mean [− 95% CI; + 95% CI]: Equatorials: 7.8 ng/ml [6.0; 10.0], Europeans: 6.0 ng/ml [3.0; 11.2]) or whether the pair was feeding-young (Equatorials: 10.4 ng/ml [8.3; 13.0], Europeans 5.6 ng/ml [3.2; 9.3]). Breeding stage did not have a significant influence (F1,54 = 2.005, p = 0.163; r 2 = 0.15). Discussion In this study, we showed that stonechats from equatorial Kenya express high levels of testosterone during the nestbuilding and laying stage and low levels during all other breeding stages and postnuptial molt. Consistent with the previous study by Dittami and Gwinner (1985), we found the highest levels of testosterone during nest-building and laying. With an average of 2.8 ng/ml, the levels of the current study were approximately 1 ng/ml higher than the levels reported by Dittami and Gwinner (1985). During all other breeding stages and during postnuptial molt, the testosterone levels of the current study were much lower than those reported by Dittami and Gwinner (1985). They reported that the concentrations of testosterone remained close to or above 1 ng/ml year-round, but the breeding stage of most birds caught was not known. Our study suggests that equatorial stonechats have a single short and high peak of testosterone during nest-building and laying, levels of far less than 1 ng/ml during all other substages of the breeding life-history stage, and undetectable levels during molt. We do not know why the pattern of testosterone levels differs between the Dittami and Gwinner (1985) and our current study. Population differences are unlikely, as the two study sites are only few kilometers apart. One explanation might be that Dittami and Gwinner (1985) conducted their study during two exceptionally dry years (J. Dittami, personal communication), perhaps explaining the lower levels during the nest-building and laying stage and the higher levels in other stages. The pattern we found in equatorial stonechats was in part similar to that of most other tropical birds with year-round territoriality: they express low levels of testosterone during most of the year (Levin and Wingfield, 1992; Hau et al., 2000; Wikelski et al., 2003; Day et al., 2006; Fedy and Stutchbury, 2006). In contrast to these other tropical birds, however, equatorial stonechats showed a pronounced and high peak of testosterone during the period of nest-building, i.e. when females are receptive and eggs are fertilized. This peak in testosterone may either be triggered by increased male–male competition or by courtship including mate-guarding and female sexual behavior (Harding, 1981; Moore, 1982, 1983; Wingfield and Farner, 1993; Wingfield and Silverin, 2002; Wiley and Goldizen, 2003). Possibly, nest-building was the most demanding stage for male stonechats, because their corticosterone concentrations peaked during this breeding stage as well. Although the corticosterone concentrations we measured cannot be considered baseline (Romero and Reed, 2005), the levels we found were similar to the baseline levels collected during an earlier field study of equatorial stonechats in Tanzania (Scheuerlein et al., 2001). In this earlier study, males that had to share their territory with a fiscal shrike (Lanius collaris), a predator of small passerine birds and their nestlings,
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had higher corticosterone concentrations than males in territories without shrikes (Scheuerlein et al., 2001). The corticosterone concentrations of Kenyan males in the nestbuilding and laying stage were similar to those of Tanzanian males cohabiting with a shrike. This similarity renders further support for the idea that the nest-building and laying stage, during which the eggs were fertilized, was a demanding stage for males. Our birds were either caught with food-baited traps or by placing a mounted decoy into the territory. Overall, the pattern of testosterone and corticosterone secretion did not differ between capture methods, suggesting that the major determinant of testosterone and corticosterone secretion was the lifehistory stage and not the capture method. Interestingly, within the group that was caught with a decoy, testosterone concentrations decreased as the exposure time to the decoy increased. In contrast, corticosterone concentrations increased with exposure time. What does that mean? There are two possible explanations: First, testosterone may decrease and corticosterone may increase during a territorial challenge. Similar patterns have been found in northern pintails (Anas acuta), great tits (Parus major) and blue tits (Cyanistes caeruleus; Sorenson et al., 1997; Van Duyse et al., 2004; M. Landys, W. Goymann, M. Raess and T Slagsvold, unpublished data). Second, individuals with high testosterone and low corticosterone levels may be more likely to detect a decoy and react faster to it than individuals expressing low testosterone and high corticosterone levels. In equatorial stonechats, the latter explanation is more likely, as we did not find a relationship between testosterone or corticosterone and the time between attacking the decoy and capture. However, most birds were caught within less than 3 min after they had attacked the mounted decoy and this time may not have been long enough to mount a hormonal response (Wingfield and Wada, 1989). Tropical and temperate comparison Testosterone concentrations of free-ranging equatorial stonechats were significantly lower than those of northern-temperate stonechats during all breeding stages, except during the nestbuilding and laying stage. In fact, concentrations of testosterone during the nest-building and laying stage tended to be higher in equatorial stonechats than in their northern-temperate relatives. If we had included data for second and third broods of European stonechats, this difference during nest-building and laying would have been significant, because testosterone concentrations of European stonechats during nest-building and laying are lower during second and third broods compared to the first brood of the year. These results are in line with the idea that tropical birds generally have lower levels of testosterone than northern-temperate species (see references in the introduction and review in Stutchbury and Morton, 2001). Equatorial stonechats are territorial year-round and hence, testosterone may play only a minor role for territory establishment and maintenance. Our results also support the hypothesis that highly seasonal breeding is associated with high peak levels of
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testosterone at both low and high latitudes in stonechats. This is in accordance with the comparative study of Goymann et al. (2004) who found that the length of the breeding season is the major determinant of peak testosterone concentrations in tropical birds: the shorter the breeding season, the higher the peak levels of testosterone. Pairs of equatorial stonechats raise only one clutch per breeding season, which takes them less than 3 months. Hence, their breeding season is shorter than that of European stonechats which raise 2–3 clutches in a period of roughly 5 months. The results of this study suggest that equatorial stonechats belong to the group of tropical species, that combine a short breeding season with high peak levels of testosterone (Goymann et al., 2004). Because peak levels of testosterone coincide with the period of maximum fertility of the female, the pronounced increase in testosterone may either be stimulated by receptive behavior of the female or by increased risk of extra-pair behavior during this critical period. Within a taxon, similar results have been found by Moore et al. (2002) on birds of the genus Zonotrichia: during the breeding season equatorial rufous-collared sparrows (Zonotrichia capensis) mounted a higher testosterone response to GnRH injections than arctic Gambel's white-crowned sparrows (Z. leucophrys gambelii) and northern-temperate Puget Sound white-crowned sparrows (Z. leucophrys pugetensis). Possibly, there is also a phylogenetic aspect to the magnitude of testosterone levels in tropical birds. Testosterone patterns of tropical birds may differ between species with a phylogenetic origin in the temperate zones and species with a tropical phylogenetic history, i.e. tropical birds originating in temperate regions may express higher levels of testosterone than those with a true tropical phylogenetic history. Tentative evidence suggests that stonechats originated in the Eurasian tropics with an African branch diverging from this original line (Wink et al., 2002a,b; M. Wink, personal communication). Hence, stonechats are more likely of tropical than of temperate origin. For the rufous-collared sparrow, it seems to be unresolved whether it is of tropical or southern temperate origin (Zink and Blackwell, 1996, I. T. Moore, personal communication). Spotted antbirds (Hylophylax n. naevioides), another yearround territorial tropical bird species, are considered to have evolved in the tropics, but also they express brief periods with high levels of testosterone (Canoine et al., in press), suggesting that the phylogenetic history may be of only minor importance. We have previously measured testosterone metabolite concentrations from droppings of several stonechat populations in captivity (Roedl et al., 2004). Captive male equatorial stonechats kept individually in cages where they could hear but not see each other expressed a seasonal pattern in testosterone metabolite concentrations with higher levels during the breeding season and lower levels thereafter (Roedl et al., 2004). These captive equatorial stonechats generally had lower levels of testosterone metabolites than European stonechats kept under similar conditions. However, they did not show a pronounced peak in testosterone metabolite concentrations similar to the plasma testosterone peak that free-living conspecifics showed during the nest-building and laying stage. We may have missed this peak in the captive study
because we sampled birds in intervals of 4 weeks only. Alternatively, the peak may result from supplementary environmental cues (i.e. interactions with females or other males). Although tropical birds including equatorial stonechats respond to photoperiod (Gwinner and Dittami, 1985; Hau et al., 1998), it is still unclear which environmental cues drive seasonal timing of reproduction at or close to the equator where photoperiodic information is basically absent. Most likely equatorial stonechats are much more sensitive to supplementary information from the environment to time breeding and other life-history stages than their northerntemperate relatives (Gwinner and Scheuerlein, 1998; Scheuerlein and Gwinner, 2002) and hence it is conceivable that supplementary environmental cues or a female partner causes the seasonal testosterone peak. Corticosterone concentrations during the incubation and feeding stage were higher in African than in European male stonechats. Due to the limited sample size of European stonechats, these results should be considered preliminary. In conclusion, the pattern of testosterone in equatorial male stonechats follows the general pattern of many tropical birds with overall testosterone concentrations being lower than in northern-temperate birds. However, similar to other tropical birds with short breeding seasons, they express high levels of peak testosterone during the short period of maximal female fertility. Equatorial stonechats even had higher levels of testosterone during this critical period than their northerntemperate congeners. The results of this study underline the importance of carefully sampling substages of the breeding lifehistory stage. Tropical birds may express high peak levels of testosterone, but due to the often lower breeding synchrony in the tropics (Stutchbury and Morton, 2001) such brief peaks may be easily missed if the exact breeding stage is not known. Acknowledgments This paper was written after the untimely death of Prof. Eberhard Gwinner. However, the project was planned and conducted by him with assistance of some of the coauthors. Therefore, Ebo Gwinner made a fundamental contribution to the writing of this paper, although he deceased before the data could be analyzed. We thank Nicole Geberzahn and Barbara Helm and two anonymous referees for reading and improving previous versions of the manuscript. Furthermore, we would like to thank Kenya Wildlife Service, the National Museum of Kenya and the Department for Ornithology for their cooperation. References Canoine, V., Fusani, L., Schlinger, B. A., Hau, M., in press. Low sex steroids, high steroid receptors: increasing the sensitivity of the nonreproductive brain. J. Neurobiol. 66. Day, L.B., McBroom, J.T., Schlinger, B.A., 2006. Testosterone increases display behaviors but does not stimulate growth of adult plumage in male goldencollared manakins (Manacus vitellinus). Horm. Behav. 49, 223–232. Dittami, J.P., 1986. Seasonal reproduction, molt and their endocrine correlates in two tropical Ploceidae species. J. Comp. Physiol., B 156, 641–647.
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