Seasonal and social modulation of testosterone in Costa Rican rufous-collared sparrows (Zonotrichia capensis costaricensis)

General and Comparative Endocrinology 166 (2010) 581–589

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Seasonal and social modulation of testosterone in Costa Rican rufous-collared sparrows (Zonotrichia capensis costaricensis) Elizabeth A. Addis a,*, D. Shallin Busch a,b, Aaron D. Clark a, John C. Wingfield a,c a

Department of Biology, University of Washington, Seattle, WA 98195, USA Northwest Fisheries Science Center, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd. E., Seattle, WA 98112, USA c Department of Neurobiology, Physiology and Behavior, University of California – Davis, 1 Shields Ave., Davis, CA 95616, USA b

a r t i c l e

i n f o

Article history: Received 3 September 2009 Revised 2 January 2010 Accepted 8 January 2010 Available online 13 January 2010 Keywords: Testosterone Zonotrichia capensis Tropics Reproductive synchrony Reproduction Social modulation

a b s t r a c t Previous work shows that most birds breeding in northern temperate regions adjust production of testosterone in response to stage of the breeding cycle and in some cases following social interactions. In contrast, prior research suggests that tropical breeding birds are less likely to modulate testosterone in response to social interactions (the propensity to increase testosterone in response to social instability is known as the challenge hypothesis). To further test the challenge hypothesis in tropical birds, we investigated whether variation in season affects reproductive condition, aggressive behavior, and social modulation of testosterone in two populations of Costa Rican rufous-collared sparrow, Zonotrichia capensis costaricensis. We conducted our study at three distinct times of year: the dry season (March and May); the veranillo, a hiatus in the rainy season (July); and the late rainy season (November). Significantly more birds were in breeding condition in the dry season than in the rainy season or veranillo. In each time period, we collected baseline testosterone samples and conducted simulated territorial intrusions (STIs). Our study shows that testosterone is modulated with season independent of breeding condition, as testosterone levels were affected by season, breeding condition, and an interaction of the two factors. Males breeding in the dry season had higher plasma testosterone levels than non-breeding males in the dry season and both breeding and non-breeding males in the veranillo and rainy season. Males did not socially modulate testosterone in any season. Aggressive behaviors expressed during STIs did not differ among seasons with the exception that males sang fewer songs during the rainy season. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction The androgen hormone testosterone is essential to male reproduction. In most vertebrates testosterone has pleiotropic effects on male reproductive behavior, secondary sexual characteristics, muscle hypertrophy, and sperm production (Balthazart, 1983; Brown and Follett, 1977; Hirschenhauser and Oliveira, 2006; Wingfield and Farner, 1993; Wingfield et al., 2000). However, testosterone also has been shown to have deleterious effects on health. Sustained elevated levels of testosterone can compromise immune function (Casto et al., 2001), increase predation risk (Dufty, 1989), inhibit parental care (Lynn, 2008), decrease fat stores, and increase risks of injury through aggressive encounters (Hau, 2007; Wingfield et al., 2001). Evidence suggests that many vertebrate species modulate testosterone levels to gain its beneficial effects, but minimize its detrimental effects (Buck and Barnes, 2003; Creel et al., 1993; Hau, 2007; Hirschenhauser and Oliveira, 2006; Wingfield et al., 1990). * Corresponding author. Fax: +1 206 543 3041. E-mail address: [email protected] (E.A. Addis). 0016-6480/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2010.01.011

In many avian species, especially those breeding in temperate habitats, testosterone levels are elevated at times of territory establishment and mate recruitment (Goymann et al., 2007; Hunt et al., 1997; Ketterson et al., 1992; Wingfield et al., 1990). During these periods, male–male competition is strong and often leads to increased aggression frequently mediated by testosterone (Hirschenhauser and Oliveira, 2006; Wingfield and Hahn, 1994; Wingfield et al., 1990). When the breeding season is short, nesting attempts tend to be more synchronous (Benson and Winker, 2001; Garamszegi et al., 2008; Hemborg et al., 2001). Short, synchronous breeding seasons intensify the pressure for territory defense and mate acquisition. Garamszegi et al. (2008) found that this intensification correlates positively with testosterone levels in a range of avian species. The fluctuation of testosterone levels according to life history stage is known as seasonal modulation of testosterone (Goymann, 2009; Goymann et al., 2007). In addition to synchronicity and brevity of breeding tending to correlate with higher testosterone, latitude does as well (Garamszegi et al., 2008). In addition to modulation of testosterone with life history stages, it can also occur rapidly in response to social cues. For instance, species with longer, asynchronous breeding seasons

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tend to have more flexibility over whether they acutely adjust circulating testosterone titers (Garamszegi et al., 2008). Some breeding populations that have multiple clutches acutely elevate testosterone levels in response to social instability and the presence of a reproductively receptive female throughout the breeding season (Goymann, 2009; Goymann et al., 2007). This acute increase is known as social modulation of testosterone, or more precisely as the ‘‘challenge hypothesis” (Wingfield et al., 1990). Some species even maintain constant levels of testosterone throughout the breeding season (Goymann, 2009; Goymann et al., 2007). We are interested in parsing apart how testosterone titers and aggressive behaviors are affected by seasonal factors and life history state. We did so by examining a system in which breeding is asynchronous within and among seasons. In a comprehensive comparative study, Goymann et al. (2004) proposed such interactions, and in this study we empirically test them. The ability to socially modulate testosterone also may be influenced by the species’ type of mating system (Hirschenhauser and Oliveira, 2006; Hirschenhauser et al., 2003). Social modulation of testosterone may be flexible in species in which paternal care is not essential, likely because testosterone can be inhibitory to parental behavior (Lynn, 2008). In populations with mandatory paternal care, males may become insensitive to testosterone during the parental period (Lynn, 2008; Lynn et al., 2005, 2009). Many tropical bird species have long breeding seasons, exhibit asynchronous breeding (Goymann et al., 2007; Stutchbury and Morton, 2001), and have low levels of testosterone year round (Goymann et al., 2004, 2007; Hau et al., 2008; Hau, 2007; Wikelski et al., 2003). Some tropical species, like the spotted antbird (Hylophylax naevioides) do socially modulation testosterone (Wikelski et al., 1999). However, other species, do not (Gill et al., 2008; Wingfield and Lewis, 1993; Moore et al. 2004a). For example, unlike some socially monogamous species of mid to high latitudes (Goymann, 2009; Goymann et al., 2007; Hirschenhauser et al., 2003), the rufous-collared sparrow (Zonotrichia capensis costaricensis) in Ecuador expresses high levels of testosterone throughout a lengthy (five month), but distinct, breeding season, and shows no social modulation of testosterone (Moore et al., 2004a). We explore the role of seasonal and social cues on testosterone titers and territorial aggression in males from two populations of the rufous-collared sparrow, Z. c. costaricensis, in Costa Rica. Unlike in Ecuador where the birds have a definitive breeding period (Moore et al., 2004a), populations of Z. c. costaricensis in Costa Rica are highly asynchronous in expression of breeding but have two loosely defined breeding periods (Wolf, 1969). The breeding periods are not completely in alignment with Costa Rica’s two primary seasons, the rainy and the dry. Because of this variability, we were able to examine the effect of season on patterns of testosterone secretion regardless of life history stage in the Costa Rican populations in a way impossible in the Ecuadorian populations (Moore et al., 2004a). We hypothesize that annual seasonal variation will influence testosterone levels and aggressive behaviors in an asynchronously breeding species. Building upon this hypothesis, we test two predictions: (1) Season impacts breeding condition, testosterone levels, and associated territorial behavior; and (2) Z. c. costaricensis do not socially modulate testosterone, like Z. c. costaricensis found in Ecuador. To do so, we examined the correlation between breeding condition, testosterone levels and territorial behavior during the dry season, a dry period in the midst of the rainy season, and the rainy season. To determine if territorial interactions between males affect testosterone titers differently over the annual cycle, we conducted simulated territorial intrusions (STIs) during those three periods as well.

2. Methods 2.1. Study population and study area Zonotrichia capensis is an emberizine sparrow that is basal to the genus Zonotrichia (Zink and Blackwell, 1996). The species ranges from Chiapas, Mexico to the southern tip of South America, and, in the tropics, is found mostly in montane areas (Chapman, 1940; Stiles and Skutch, 1989). Z. capensis is socially monogamous and both males and females exhibit parental care (Chapman, 1940; Miller and Miller 1968). Most birds synchronize reproductive capacity only with their mate and not the whole population (Miller and Miller, 1968; Moore et al., 2005; Wolf, 1969). In addition to territory holders, resident populations of Z. capensis can also have ‘‘floaters” – birds that do not possess territories and move among multiple territories belonging to others (Smith, 1978). Floaters can take over other male’s or abandoned territories when the opportunity presents itself (Busch et al., 2004; Smith, 1978). We conducted this study on the subspecies Z. c. costaricensis at two montane sites in Costa Rica: Cuerici Biological Station (N 09° 330 13.90 0 , W 83°400 04.100 ; 2585 m) and Finca dos Lados (10°100 8.40 0 , W 84°170 2.000 ; 1780 m). We collected samples over the course of four field excursions in an effort to encompass the three major seasons at our field sites: (1) dry season, 9 April–3 May 2005 and 12–23 March 2006; (2) dry period in the middle of the wet season (termed veranillo (Janzen, 1983)), 11–25 July 2004; and (3) rainy season, 5–15 November 2007. During all study periods, birds were abundant at both sites in pastures and open farmland. No birds were sampled more than once. Specific ages of the study subjects were not known, but all sampled birds were adults, based upon plumage. 2.2. Breeding condition and sex determination We determined the sex of the subjects genetically if the bird was not in breeding condition and by examination of external anatomy if the individual was in breeding condition. We extracted DNA from red blood cells with washes of 70% ethanol or a Qiagen DNEasy tissue kit. For genetic sex determination, we followed a modified protocol of Griffiths et al. (1998). Briefly, we used polymerase chain reaction (PCR) to amplify sections of the CHD-W gene (unique to females) and the CHD-Z gene (found in both females to males) using primers P2 and P4. After PCR amplification, the amplified gene fragments were run on a 2.5% agarose gel. Because the size of the amplified gene fragments differed between males and females, we were able to determine sex by visual examination of the gel (females had two bands, males only one). We visually examined the cloacal protuberance (CP) in males and brood patch in females to assign sex and breeding state if the individual was in breeding condition. Males were classified as breeding if either the width or the height of the CP was greater than 5 mm (Miller, 1959a). If the brood patch region of the female was defeathered, edematous, bare and wrinkled, or re-feathering we considered the female to be in breeding condition. Females without brood patches were classified as non-breading. 2.3. Simulated territorial intrusions To determine if territorial interactions between males affects testosterone titers differently over the annual cycle, we conducted simulated territorial intrusions (STIs) in the dry season, the veranillo, and the rainy season. Territorial males were identified by their response to playback of conspecific song. Once we selected a responsive male, we used the STI protocol described by Wingfield and Hahn (1994). We placed a conspecific decoy adjacent to a furled mistnet along with a playback speaker. We played conspe-

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cific song for 10 min, during which time we collected data on the following behaviors: (1) closest approach of the focal bird to the decoy; (2) time the focal bird spent within five meters of the decoy; (3) number of songs of the focal bird; and (4) number of flights of the focal bird. After 10 min, we turned off the playback, opened the net, and resumed the playback until the focal bird was captured or for 45 min, after which the STI was aborted. In temperate Z. leucophrys, a 10 min STI sufficient for testosterone levels to increase (Wingfield and Hahn, 1994). Z. c. costaricensis in Ecuador show no difference in testosterone levels among STIs that are 10, 30, or 60 min (Moore et al., 2004a). After capture, we took a blood sample from the focal bird. Plasma testosterone levels after the STI were compared with those from male caught passively either in mist nets or potter traps baited with crushed corn. At each site, we recorded songs of three individual males to use as playback during the STIs. The playback consisted of one song followed by 10 s of silence. This sequence was then looped so the playback could be repeated indefinitely. At each site, we randomly used one of three local recordings during STIs to avoid pseudoreplication (Kroodsma et al., 2001). 2.4. Blood sampling and hormone analysis Blood was collected from the alar vein of each sampled bird within 10 min of capture using a 26 gauge needle and heparinized microhematocrit tubes. Each blood sample was about 250 ll, approximately 1% of the bird’s mass. Samples were stored on ice until the end of the day, when they were centrifuged to separate the plasma from red blood cells. The plasma was aspirated and frozen until it could be assayed. The red bloods cells were frozen and saved for sexing via genetic analysis. Testosterone concentrations were determined using a direct radioimmunoassay (see Wingfield et al. (1991)) with an antibody that binds to primarily testosterone and secondarily dihydrotestosterone. Additionally, dihydrotestosterone levels tend to parallel testosterone levels in songbirds. Therefore, we will refer to hormones measured simply as ‘‘testosterone.” In brief, 2000 cpm of tritiated testosterone were added to all samples to determine the efficiency of hormone extraction from the plasma. Testosterone was extracted from the plasma with dichloromethane. The dichloromethane was then aspirated and evaporated under a stream of nitrogen at 45 °C, and the samples were re-suspended in phosphate-buffered saline with gelatin. All samples were run in duplicate, with standards to determine inter- and intra-assay variation. Plasma volumes of the samples varied from 50 to 200 ll and the detection limit of the assay ranged from 0.05 to 0.09 ng/ml, depending on the volume of the plasma samples. A total of three assays were run for all of the samples with intra- and inter-assay variation of 11% and 16.8%, respectively. 2.5. Statistical analysis To determine if reproductive condition varied with season, we used a chi-squared test comparing the number of males and females in breeding and non-breeding condition (N = 221; the sample size for this comparison incorporated birds sampled for Busch et al. (2010)). We used least squares mean models to explore the impact of season, reproductive condition, and STI treatment on testosterone levels. All testosterone concentrations were log-transformed to meet normality requirements before statistical analysis, verified using a Shapiro–Wilk goodness-of-fit test. We built least squares mean models with all variables and all interactions and then removed non-significant terms in a step-wise manner until only significant terms remained. We selected the best least squares

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mean model using the Akaike Information Criterion corrected for small sample sizes (AICc). The model with the lowest AICc value was considered the best-fit model (Burnham and Anderson, 1998). We used Tukey-HSD tests for post-hoc analysis to distinguish among groups with significant terms in the least squares mean models. We also compared variance of testosterone levels between breeding and non-breeding birds using a Levene’s test. The a level for significance was set at p 6 0.05. Data in figures represent the arithmetic mean plus standard error. The behavioral data collected during the STIs were combined using a principal component analysis (PCA). The first principle component of the PCA (PC1) was used as a score of composite aggression. We also conducted a one-at-a-time (OAT) analysis to determine if the exclusion of behaviors would increase the percent variance explained. We tested for significant relationships between season and aggression and for an interaction between aggression and season using a two-way ANOVA. Additionally, we compared the composite aggression score between focal males that were caught after the STI and males that we were unable to catch post-STI. We used a Tukey-HSD test for post-hoc analysis. Importantly, behaviors were measured in the same manner for birds that were caught and those that were not. Both males that were caught and those that were not were present for the entire duration of the STI. Because there could be an interaction between breeding state and behavior, we ran analyses on behavioral data twice: once with both breeding and non-breeding males both caught and uncaught and again using only caught, breeding males. As the results of the statistical analyses were the same for both data sets, we discuss only the results from the analysis on both breeding and non-breeding birds. Additionally, we ran a linear regression between log-transformed testosterone and all behaviors to determine if a correlation existed between them. All statistical analyses were conducted in JMP version 7 (SAS Institute, 2007).

3. Results 3.1. Season Reproductive condition of both males and females varied significantly with season (Fig. 1). In both sexes, more birds were breeding in the dry season than in the veranillo or rainy seasons (v2 = 66.64, p < 0.0001). The best-fit least squares mean model indicated that breeding state, season, and an interaction between breeding state and season explained a significant amount of variation in testosterone levels (model: F5,90 = 25.16, p < 0.0001; breeding: F1,90 = 31.85, p < 0.000 l; season: F2,90 = 12.86, p = 0.002; interaction effect between breeding and season: F2,90 = 14.56, p = 0.001) (Table 1 and Fig. 2). Testosterone levels were higher in breeding than nonbreeding males in both the dry season and veranillo, but not in the rainy season. Additionally, testosterone levels in breeding birds were higher in the dry season than in the veranillo and rainy season (Fig. 2). The variance of testosterone levels did not differ between breeding and non-breeding males (Levene’s test: F2,62 = 1.38, p = 0.23).

3.2. Simulated territorial intrusions 3.2.1. Testosterone Circulating testosterone titers in breeding birds did not change with STI treatment (model: F5,64 = 8.93, p < 0.0001; Treatment: F1,64 = 0.031, p = 0.861) (Fig. 3), but again, testosterone titers were higher in the dry season than in the veranillo or rainy season (season: F2,64 = 24.88, p < 0.0001) (Fig. 2).

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100%

Female Male

58

Percent breeding

42

80% 60% 51

26

40% 21

20%

23

0% Veranillo

Dry

Rainy

Breeding season Fig. 1. Percent of birds caught in breeding condition. Sample sizes are given on column tops. Season had a significant effect on breeding condition.

Table 1 Results of least squares mean models on plasma T concentrations. All models fit the data significantly. The model in bold is considered the best-fit because it has the lowest AICc score. Model Season, Breeding, STI, Season  Breeding, Season, Breeding, STI, Season  Breeding, Season, Breeding, STI, Season  Breeding, Season, Breeding, STI, Season  Breeding, Season, Breeding, STI, Season  Breeding Season, Breeding, Season  Breeding

4.5

Season  STI, Breeding  STI, Season  Breeding  STI Season  STI, Breeding  STI Season  STI Breeding  STI

a

Testosterone (ng/ml)

4

AICc

DAICc

r2

Significant factors

189.06 188.76 186.56 185.34 185.41 183.18

5.87 5.58 3.38 2.16 2.23 0

0.58 0.57 0.57 0.56 0.57 0.57

Season, Breeding, Season  Breeding Season, Breeding, Season  Breeding Season, Breeding, Season  Breeding Season, Breeding Season, Breeding, Season  Breeding Season, Breeding, Season  Breeding

Breeding Not breeding

36

3.5 3 2.5

b

2

20

b,c

1.5 1

b,c

4

0.5

c

12

c 9

10

0

Veranillo

Dry

Rainy

Season Fig. 2. Testosterone levels of breeding and non-breeding males sorted by season. Testosterone levels varied significantly with season and breeding condition. Significant differences are denoted by different letters. Sample sizes are given on column tops.

4.5

Testosterone (ng/ml)

4

a 20

Baseline STI

a

3.5 20

3

a,b

2.5 2

11

b

1.5 1

10

b

b 19

0.5

11

0 Dry

Veranillo

Rainy

Season Fig. 3. Baseline and post-STI testosterone levels of breeding males sorted by season. Testosterone levels varied significantly with season, but not STI. Significant differences are denoted by different letters. Sample sizes are given on column tops.

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3.2.2. Behavior PC1 explained 50% of the variance in behaviors, an amount typical for this species (Moore et al., 2004a, 2004b) (Table 2). From our OAT analysis, we found that by excluding the number of songs from the PCA, the percent of variance explained by PC1 increased to 58%. However, as the final results did not differ whether we classified aggressive behavior as including songs or not, we only report the results of the initial analysis based upon the PC1 that included all behaviors. Aggressive behavior varied only between males that were caught post-STI and males that were not (model: F5,62 = 7.28, p < 0.0001; caught status: F1,62 = 17.68, p < 0.0001) and not with season (F1,62 = 1.16, p = 0.32) or an interaction between season and caught status (F1,62 = 0.16, p = 0.85). To explore which components of behavior varied between males caught and not caught, we tested each behavioral constituent for significant differences among groups (Fig. 4). Males that were caught flew significantly more times (model: F5,68 = 2.86, p = 0.02; capture status: F1,68 = 5.83, p = 0.02), approached closer to the decoy (model: F5,68 = 4.26, p = 0.002; capture status: F1,68 = 8.96, p = 0.004) and spent more time within 5 m of the decoy (model: F5,62 = 4.05, p = 0.003; capture status: F1,62 = 14.41, p = 0.0004) than males that were not caught. There was no difference in the number of songs sung between males that were caught and those that were not, but the number of songs varied with season. Males sang significantly less in the rainy season than in the dry season or veranillo (model: F5,68 = 7.50, p < 0.0001; season: F2,68 = 7.16, p = 0.002). Other behavioral measures did not change significantly with season (flights: F2,68 = 0.42, p = 0.66; closest approach: F2,68 = 0.81, p = 0.45; time within 5 m: F2,62 = 0.43, p = 0.65). A weak correlation did exist between plasma testosterone titers and the number of songs a male sang (F1,43 = 12.6, p = 0.001; r2 = 0.21), although not for the other behaviors (number of flights: F1,43 = 0.072, p = 0.79; closest approach; F1,43 = 0.60, p = 0.44; time within 5 m: F1,39 = 0.003, p = 0.96). 4. Discussion We explored the role of season, breeding condition, and social interactions on plasma levels of testosterone to test the challenge hypothesis and the factors that control testosterone titers in asynchronously breeding populations of Costa Rican Z. capensis. Similar to most birds studied to date reproductive condition at the population level varied significantly with season, although reproductive birds were present in all seasons. Circulating testosterone levels were higher in breeding males than non-breeding males, but also varied with season. Unlike the well-studied Z. leuocophrys and other northern temperate and arctic species (Meddle et al., 2002; Wingfield and Hahn, 1994) and some tropical species (Goymann, 2009; Goymann et al., 2007; Hau et al., 2000), circulating testosterone levels in Z. c. costaricensis did not increase in response to STI. This result concurs with Z. c. costaricensis populations in Ecuador (Moore et al., 2004a), possibly indicating that Z. c. costaricensis does not socially modulate testosterone. The ranges of circulating testosterone levels in Z. c. costaricensis in Costa Rica were within the same range as those of Ecuadorian Z. c. costaricensis, between 2.5 and 6 ng/ml (Moore et al., 2004a, 2004b). Aggression was mea-

Table 2 Loadings of principal component 1 for quantified behaviors during simulated territorial intrusions. Variable

PC1 loadings

Number of songs Number of flights Closest approach Time spent within 5 m % Variance explained

0.44 0.54 0.49 0.53 49.9

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sured by PC1 in response to an STI did not change among seasons despite changing testosterone titers. Birds that were caught postSTI were more aggressive than those that were not caught. By examining each behavioral response separately, we found that birds sang significantly less in the rainy season. Additionally, the number of songs was positively correlated with testosterone levels. 4.1. Season We found that reproductive condition varied with season, with a smaller percentage of males and females in breeding condition during the veranillo and rainy season compared to the dry season. Presence of more breeding birds in the dry season suggests that some times of year are better to breed in than others, but enough resources are available year-round for breeding to occur in some individuals at any time. A variety of proximate factors could induce the negative relationship between breeding and rain at the population level. For example, in Costa Rica seed set usually occurs in the dry season (Coen, 1983) and insect abundance peaks when the greatest number of sunny days occurs, also in the dry season (Janzen, 1983). Seed and insect bounty tend to be lowest during the rainy season (Coen, 1983; Janzen, 1983). Food abundance may be important to maximize offspring survival, driving more birds to reproduce when food is more abundant. Additionally, mean temperatures are slighter cooler in the rainy season than in the dry (Coen, 1983). An interaction between climate and thermoregulatory requirements of the birds could suppress reproduction in many individuals during the veranillo and rainy season. Photoperiod may also be a driver of reproductive condition as an ultimate cue. Captive studies have shown that Z. c. costaricensis can detect changes in photoperiods typical of higher latitude (Epple et al., 1972; Miller, 1959b, 1965). However, photoperiod annually changes by just 72 min in Costa Rica. Spotted antbirds (H. naevioides) in Panama can detect a 17-min difference in annual photoperiod and use this cue to partially entrain reproduction (Hau et al., 1998). This result suggests that it may be possible for Z. c. costaricensis in Costa Rica to cue off of small changes in day length, though the presence of breeding individuals in the population throughout the year suggests that signals from photoperiod may not affect all birds in the same way and that proximate cues, like resource abundance, may control expression of the breeding state. In this study, we were not able to determine if individuals bred more than once per year, as each bird was sampled only once. In the dry season and veranillo, breeding birds had higher testosterone levels than birds not in breeding condition. This result was expected, as testosterone is thought to be imperative to sperm production and other reproductive functions such as development and maintenance of the cloacal protuberance and song production (Smith et al., 1997). However, during the rainy season, circulating testosterone levels were very low and not different between breeding and non-breeding individuals. These results are similar to those found by Wikelski et al. (2003) in neotropical species. Of note, breeding males in the rainy season did have large cloacal protuberances, suggesting the testes are active (Birkhead et al., 1993). We do not know why circulating testosterone levels are similar between breeding and non-breeding males, but the result suggests that testosterone levels are regulated to some extent by means other than reproductive condition. As there were the fewer females in reproductive condition during the veranillo and rainy seasons, testosterone levels could be lower in males because of the low number of receptive females. A possible explanation for lower levels of testosterone during the breeding season is the proportion of birds breeding in the population. Fewer breeding birds in the population reduces access to fertile females, which may impact testosterone levels in breeders. The presence of receptive females has been shown to cause increases in

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16

a

a

a

Number of flights

14 12

b

10 8

b

6 b

4 2 0

13

27

70

Number of songs

60

11

4

a

a

2

12

a Caught

50

Not caught

40

a

30 20 10

b

b

Closest approach (m)

0 b

8

b

6 b 4 2

a

a

a

0

Time within 5m (sec)

600

a

500 400

a a

300 b

200

b

100

b

0

Dry

Veranillo

Rainy

Season Fig. 4. Behavioral responses to STI according to season, sorted by males that were caught vs. not caught. Significant differences are denoted by different letters. Sample sizes are given at column bases of top graph and are the same for all behavioral measurements.

circulating testosterone levels in male white-crowned sparrows (Goymann et al., 2007; Moore, 1983), European starlings (Pinxten et al., 2003) and song sparrows (Runfeldt and Wingfield, 1984). In equatorial stonechats, testosterone levels increase in the presence of receptive females, but not with territorial behavior (Goymann et al., 2006). A similar phenomenon is possible in Costa Rican Z. c. costaricensis. The trend of higher testosterone levels in the dry season than in the rainy season and veranillo also may be due to the density of birds in breeding condition during each season. Higher testosterone levels have been associated with higher population densities (Ball and Wingfield, 1987; Moss et al., 1994; Silverin, 1998; Wingfield and Hahn, 1994). In this instance, the size of the breeding population may be more important than the size of the actual pop-

ulation. The population density of breeding birds and access to a receptive female are likely interrelated and together promote a decrease in testosterone in males breeding during the rainy season. Observed seasonal variation in testosterone may also be linked to the function of territory for these populations of Z. c. costaricensis (Garamszegi et al., 2005; Goymann et al., 2004; Hau et al., 2008). In north temperate breeding birds, territories are generally held during the reproductive season to attract mates and extra-pair copulations (EPCs). In those populations, territory establishment and territory defense are associated with elevated levels of testosterone in the breeding season (Goymann, 2009; Goymann et al., 2007; Wingfield et al., 1990). In contrast, territories in the tropics are generally held year-round and are resource based, rather than a tool for mate recruitment (Stutchbury and Morton, 2001).

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Territory establishment tends to be opportunistic and asynchronous in the tropics, and, therefore, may not be associated with elevations in testosterone (Stutchbury and Morton, 2001). Variation in testosterone titers among seasons may be related to availability of EPCs (Garamszegi et al., 2005, 2008; Ketterson et al., 1992; Reed et al., 2006). In populations in which breeding is asynchronous, EPC rates may be low because females cannot accurately assess male quality when many males in a population are not simultaneously displaying to advertise their territory (Stutchbury and Morton, 1995), but see Weatherhead and Yezerinac (1998). However, because of the seasonal variation in breeding synchrony we observed in this study, the rate of EPCs may vary between seasons in Costa Rican Z. c. costaricensis, with more occurring during the dry season than during the veranillo and rainy season. In this study we did not look at rates of EPCs. Further studies should examine the rate of EPCs in Z. c. costaricensis in Costa Rica as an explanatory factor for testosterone levels. 4.2. Simulated territorial intrusions 4.2.1. Testosterone STIs did not trigger an increase in testosterone titers in any season, which suggests that Z. c. costaricensis do not socially modulate testosterone. This conclusion is consistent with results from the studies of Z. c. costaricensis populations in Ecuador conducted during both the breeding and non-breeding seasons (Moore et al., 2004a, 2004b). While these results do not conform to the paradigm of the challenge hypothesis based upon birds breeding in northern temperate zones, comparative analyses have shown that some species do not socially modulate testosterone, especially species that exhibit asynchronous breeding (Goymann, 2009; Goymann et al., 2004, 2007). However, alternate explanations for a lack of pronounced social modulation of testosterone are plausible. First, our results could be influenced by our emphasis on the population rather than individual level. Because at least some birds are in reproductive condition during all seasons of the year, birds could likely vary in their breeding sub-stages throughout the year. Northern temperate birds that socially modulate testosterone do so in the mid and late-breeding sub-stages when they are establishing, maintaining or re-establishing territories (Wingfield, 1994; Wingfield and Hahn, 1994; Wingfield et al., 1990). During the early breeding phase, testosterone levels tend to be maximal at all times. Later in the breeding season, during the nesting and parental phases, testosterone levels decrease, but can increase in response to STIs (Goymann et al., 2007; Wingfield, 1994; Wingfield and Hahn, 1994; Wingfield et al., 1990). It is possible that enough variation in the testosterone response to STIs occurs in the breeding sub-stages of our sampled males that any effects of STIs on testosterone levels are masked. A lack of an effect of STI on peripheral testosterone titers was also found in bay wrens (Thyrothorus nigricapillus) in Panama (Levin and Wingfield, 1992) and non-breeding song sparrows (Soma et al., 2002) (for more examples see Goymann, 2009; Goymann et al., 2007). However, if the breeding birds were caught in all sub-stages, we would expect to see greater variance in testosterone levels in breeding males than in non-breeding males, which we did not. We cannot exclude the possibility that the hormonal response of floaters affected the results, although we caught the males we believed to be the territory owner based upon behavioral observations. While historically the challenge hypothesis posited that social instability would cause changes in plasma levels of testosterone, more recent studies have shown that other components of the testosterone system can be regulated. For example, androgen and/or estrogen receptors (Ball and Balthazart, 2004; Canoine et al., 2007); aromatase, the enzyme that converts testosterone to estradiol (Ball and Balthazart, 2004; Canoine et al., 2007; Soma et al.,

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2008); or corticosterone binding globulin (CBG) (Deviche et al., 2001), a possible sex steroid binding globulin, could be regulated. None of these factors have been examined in the Costa Rican Z. c. costaricensis. However, other works makes the possibility of modulation on the variable unlikely. Moore et al. (2004b) found no changes in aggressive behavior or testosterone levels post-STI after implanting Z. capensis with flutamide (an anti-androgen) and ATD (androstatriendione, an aromatase inhibitor). In addition, Wada et al. (2006) found no correlation between testosterone and CBG levels in Z. c. costarcinesis in Ecuador, suggesting CBG is most likely not involved in social modulation of testosterone effects. Furthermore, work in blue tits (Cyanistes caeruleus) (Landys et al., 2007) and mountain white-crowned sparrows ( Z. leucophrys oriantha) (Lynn et al., 2007) found no correlations between CBG levels and testosterone post-STI. Our results do not address whether proteins associated with the regulatory actions of testosterone or other receptor co-factors or conversion enzymes (Hau, 2007) could be modulated seasonally, but this is an area for further exploration. Lastly, in species of birds in which male parental care is integral to offspring survival, males typically do not increase testosterone in response to STIs (Goymann, 2009; Lynn, 2008). While both sexes do exhibit parental care in Z. c. costaricensis (Miller and Miller, 1968), the importance of male parental care to offspring survival is unknown. The high baseline levels of testosterone in breeding birds at some times of the year would imply paternal care is not mandatory. Further studies should assess the role of male parental care in Z. c. costaricensis. 4.2.2. Behavior Using the composite score of aggression, we found a difference in aggression between birds that were caught and those that were not, but no difference in aggression among seasons. Analyzing the behaviors separately, the results followed the same trend, with the exception of one behavior: fewer songs were sung during the rainy season than the dry season, and song number did not differ between caught and uncaught birds. These results suggest that the aggressive behaviors of flight, approach to the decoy, and time spent near the decoy are not likely regulated by testosterone alone; testosterone levels fluctuate throughout the year and these behaviors do not. The correlation between low song production and lower levels of testosterone during the rainy season is consistent with the role of testosterone in activating the song production pathway (Smith et al., 1997). Similar results were found in Z. c. costaricensis in Ecuador: observations with STIs indicate that song production differs between breeding and non-breeding seasons, as does testosterone levels (Moore et al. 2004c). During the rainy season in Costa Rica, birds rarely sang spontaneously, but did sing after playback stimulus (personal observations). This result suggests that STIs do in fact trigger some form of neurological response, but the response does not affect peripheral levels of testosterone. Recent studies have shown that androgens and estrogens can be synthesized de novo in the brain (Soma et al., 2008; Tsutsui et al., 2003; Tsutsui and Yamazaki, 1995), and steroids localized in the brain could be responsible for song production. 5. Conclusion We found that testosterone levels co-vary with season and breeding condition, with testosterone levels in males breeding in the dry season being higher than testosterone levels in males breeding in the wet season independent of breeding condition. Additionally, our prediction that Costa Rican Z. c. costaricensis do not socially modulate testosterone was supported. Our data also support the prediction that season influences breeding condition, but do not support the hypothesis that season influences overall

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aggressive behavior in Z. c. costaricensis: the number of birds in breeding condition varied by season, but overall aggression did not. A notable exception was that singing did vary between seasons. In sum, the results of this study indicate that even though the Costa Rican populations of Z. c. costaricensis breed asynchronously throughout the annual cycle, seasonal variation still affects testosterone and singing behaviors. Acknowledgments We thank Sara Clark and the Solano family for kindly allowing us to work on their farms in Costa Rica (Fincos dos Lados and Cuerici Biological Station, respectively). We thank Federico Prado for help with fieldwork, and Bethanne Zelano for guidance on the genetic sexing. Francisco Campos of the Organization for Tropical Studies assisted in permit logistics. We also thank the Ministry of the Environment and Energy of the government of Costa Rica for granting us our permits. All birds were treated in accordance with the University of Washington’s IACUC protocols # 2212-24 and # 2212-46. This work was funded by the National Science Foundation (Graduate Research Fellowship to D.S.B. and research grant IBN-9905679 to J.C.W.). Two anonymous reviewers provided helpful comments that improved the paper. References Ball, G., Balthazart, J., 2004. Hormonal regulation of brain circuits mediating male sexual behavior in birds. Physiology & Behavior 83, 329–346. Ball, G.F., Wingfield, J.C., 1987. Changes in plasma levels of luteinizing hormone and sex steroid hormones in relation to multiple broodedness and nest site density in male starlings. Physiological Zoology 60, 191–199. Balthazart, J., 1983. Hormonal correlates of behavior. Avian Biology 7, 221–365. Benson, A., Winker, K., 2001. Timing of breeding range occupancy among highlatitude passerine migrants. Auk 118, 513–519. Birkhead, T.R., Briskie, J.V., Moller, A.P., 1993. Male sperm reserves and copulation frequency in birds. Behavioral Ecology and Sociobiology 32, 85–93. Brown, N., Follett, B., 1977. Effects of androgen on the testis of intact and hypophysectomized Japanese quail. General and Comparative Endocrinology 33, 267–277. Buck, C.L., Barnes, B.M., 2003. Androgen in free living arctic ground squirrels: seasonal changes and influence of staged male–male aggressive encounters. Hormones and Behavior 43, 318–326. Burnham, K., Anderson, D., 1998. Model Selection and Multimodel Inference: A Practical Information-Theoretical Approach. Springer, New York. Busch, D.S., Wingfield, J.C., Moore, I.T., 2004. Territorial aggression of a tropical passerine, Zonotrichia capensis, in response to a variety of conspecific intruders. Behaviour 141, 1173–1188. Busch, D.S., Addis, E.A., Clark, A., Wingfield, J., 2010. Disentangling the effects of environment and life-history stage on corticosterone modulation in Costa Rican rufous-collared sparrows, Zonotrichia capensis costaricensis. Physiological and Biochemical Zoology 83, 87–96. Canoine, V., Fusani, L., Schlinger, B., Hau, M., 2007. Low sex steroids, high steroid receptors: increasing the sensitivity of the nonreproductive brain. Developmental Neurobiology 67, 57–67. Casto, J.M., Nolan Jr., V., Ketterson, E.D., 2001. Steroid hormones and immune function: experimental studies in wild and captive dark-eyed juncos (Junco hyemalis). American Naturalist 157, 408–420. Chapman, F., 1940. Post-glacial history of Zonotrichia capensis. Bulletin American Museum of Natural History 77, 381–439. Coen, E., 1983. Climate. In: Janzen, D. (Ed.), Costa Rican Natural History. University of Chicago Press, Chicago, pp. 35–46. Creel, S., Wildt, D.E., Monfort, S.L., 1993. Aggression, reproduction, and androgens in wild dwarf mongooses: a test of the challenge hypothesis. American Naturalist 141, 816–825. Deviche, P., Breuner, C.W., Orchinik, M., 2001. Testosterone, corticosterone, and photoperiod interact to regulate plasma levels of binding globulin and free steroid in dark-eyed juncos, Junco hyemalis. General and Comparative Endocrinology 122, 67–77. Dufty, A.J., 1989. Testosterone and survival: a cost of aggressiveness? Hormones and Behavior 23, 185–193. Epple, A., Orion, G.H., Farner, D.S., Lewis, R.A., 1972. The photoperiodic testicular response of a tropical finch, Zonotrichia capensis costaricensis. Condor 74, 1–4. Garamszegi, L., Eens, M., Hurtrez-Bousses, S., Moller, A.P., 2005. Testosterone, testes size, and mating success in birds: a comparative study. Hormones and Behavior 47, 389–409. Garamszegi, L., Hirschenhauser, K., Bokony, V., Eens, M., Hurtrez-Bousses, S., Moller, A.P., Oliveira, R.F., Wingfield, J.C., 2008. Latitudinal distribution, migration, and testosterone levels in birds. American Naturalist 172, 534–546.

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