Sex ratio and male sexual characters in a population of blue tits, Parus caeruleus

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1 Behavioral Ecology doi: /beheco/ari089 Advance Access publication 12 October 2005 Sex ratio and male sexual characters in a population of blue tits, Parus caeruleus A. Dreiss, a M. Richard, a F. Moyen, b J. White, a A.P. Møller, c and E. Danchin d a Laboratoire Fonctionnement et Evolution des Systèmes Ecologiques, UMR 7625, Université Pierre et Marie Curie, Bât. A, Case 237, 7ème étage, 7 quai Saint-Bernard, Paris Cedex 5, France, b Laboratoire d Ecologie Générale, Muséum d Histoire Naturelle, Brunoy, France, c Laboratoire de Parasitologie Evolutive, Université Pierre et Marie Curie, Paris, France, and d Laboratoire Evolution et Diversité Biologique, Université Paul Sabatier, Toulouse, France Sex allocation theory proposes that parents should bias the sex ratio of their offspring if the reproductive value of one sex is greater than that of the other. In the monogamous blue tit (Parus caeruleus), males have a greater variance in reproductive success than females, and high-quality males have higher reproductive success than high-quality females due to extrapair paternity. Consequently, females mating with attractive males are expected to produce broods biased toward sons, as sons benefit more than daughters from inheriting their father s characteristics. Song and plumage color in birds are secondary sexual characters indicating male quality and involved in female choice. We used these male sexual traits in blue tits to investigate adaptive sex ratio manipulation by females. We did not find any relationship between male color ornamentation and brood sex ratio, contrary to previous studies. On the other hand, the length of the strophe bout (i.e., the mean number of strophes per strophe bout) of fathers was positively related with the proportion of sons in their broods. The length of the strophe bout is supposed to reflect male quality in terms of neuromuscular performance. We further showed that sons produced in experimentally enlarged broods had shorter strophe bouts than sons raised in reduced broods. These results are consistent with the hypothesis that females adjust the sex ratio of their broods in response to the phenotype of their mate. Key words: dawn chorus, male song, Parus caeruleus, plumage color, sex ratio. [Behav Ecol 17:13 19 (2006)] According to sex allocation theory, if parents could predict the relative reproductive value of their sons and daughters, individuals that are able to bias the sex ratio of their offspring toward the sex with the higher reproductive value would be favored by natural selection (Trivers and Willard, 1973). Trivers and Willard (1973) suggested that parental characteristics and condition, as factors determining offspring quality, are likely to be essential factors determining the fitness costs and benefits of overproducing sons or daughters. Parents that optimize the sex ratio of their brood accordingly should be selectively favored. Although the mechanism is unknown, certain parental characteristics, like male survival (Sheldon, 1999) and female body condition (Nager et al., 1999), have been found to be linked to an adaptive skew in brood sex ratio. Sexual attractiveness of a male may hypothetically affect the optimal sex ratio of his brood(s). If male attractiveness is transmitted to his sons, then sons of a highly attractive male may be of greater reproductive value than daughters. Conversely, daughters of unattractive males may be of relatively higher reproductive value than sons. If that were the case, it would be adaptive for both parents to bias the sex ratio in response to male secondary sexual characters. In birds, certain studies have shown evidence of adaptive bias (reviewed in Cockburn et al., 2002), although there is no evidence of adaptive bias across all studies conducted so far (Ewen et al., 2004). Within species and populations, great variability exists in sex ratios. For instance, in the collared flycatcher (Ficedula albicollis), brood sex ratio is Address correspondence to A. Dreiss. adreiss@snv.jussieu.fr. Received 27 October 2004; revised 25 July 2005; accepted 31 August Ó The Author Published by Oxford University Press on behalf of the International Society for Behavioral Ecology. All rights reserved. Downloaded from For permissions, please journals.permissions@oxfordjournals.org male biased when the male rearing the brood has a large forehead patch (Ellegren et al., 1996). This trait is a heritable secondary sexual character involved in female choice (Sheldon et al., 1997) and related to male condition (Gustafsson et al., 1995). However, other studies did not find such a trend. For instance, in the barn swallow (Hirundo rustica), females do not seem to adjust sex ratio in relation to their mate s tail length (Saino et al., 1999), a character that is involved in female choice (Møller, 1995) and which appears to signal viability (Møller, 1994). However, when the sex ratios produced by the same females mated in different years to males with different tail lengths were recorded, there was a strong positive relationship between difference in tail length of the two males and difference in brood sex ratio (Saino et al., 2002). This result emphasizes the importance of controlling for potentially confounding variables when attempting to analyze factors hypothesized to affect brood sex ratios. We studied brood sex ratios and their relationship with male sexual characters, that is to say male song and coloration, in the blue tit (Parus caeruleus), a socially monogamous passerine. Previous studies have shown that females mated to males with high survival prospects (Svensson and Nilsson, 1996) or with a plumage with high UV reflectance (Sheldon et al., 1999) bias hatching sex ratio of their broods in favor of sons. Another study on the great tit (Parus major) found a correlation between brood sex ratio and male body size (Kölliker et al., 1999). However, findings are inconsistent among blue tit populations because Leech et al. (2001) found no significant correlation between sex ratio and parental quality as reflected by biometrics or apparent parental survival in a study based on very large sample sizes. There are no studies of the relationship between song and offspring sex ratio in blue tits or any other species. Song in birds is known to be a secondary sexual character reliably reflecting male quality and involved in female choice (Andersson,

2 14 Behavioral Ecology 1994). Therefore, individual variation in song characteristics affects male reproductive success. Indeed, male blue tits that produce extrapair offspring have longer strophes (song units constituted of an uninterrupted succession of sounds and separated from other units by a pause) than males that lose paternity (Kempenaers et al., 1997). Furthermore, individual variation in song production depends on male quality. Tarsus length, an indicator of rearing conditions, is linked to song repertoire size in blue tits (Doutrelant et al., 2000). Strophe duration could also indicate male quality in blue tits because Bijnens (1988) found a correlation between strophe duration and apparent survival rate. Finally, song constitutes a costly signal and consequently is believed to represent an honest signal of quality (Gil and Gahr, 2002). In this study, we determined whether natural variation in brood sex ratio in a population of blue tits was related to song characteristics of the attending male during the dawn chorus. If song is a signal used by the female to assess mate quality, we predicted that females mated with males producing highquality songs should bias their broods toward sons. Furthermore, we aimed to investigate whether the natural crown color, a sexual signal showing sexual dimorphism (Andersson et al., 1998; Hunt et al., 1998), is related to sex ratio, as found experimentally by Griffith et al. (2003) and Sheldon et al. (1999). We also tested whether the sex ratio of extrapair chicks was male biased, as predicted by theory. Indeed, it is commonly assumed that extrapair males are of higher quality than social partners (Kempenaers et al., 1992), and thus extrapair young may have higher reproductive value than within-pair young. Nevertheless, recent studies of birds did not find evidence consistent with such a relationship (Leech et al., 2001; Saino et al., 1999; Sheldon and Ellegren, 1996; Westerdahl et al., 1997). Bird song is a complex trait that is affected not only by factors such as predation, aggressiveness, environmental quality, and ability to raise an immune response (Gil and Gahr, 2002) but also by developmental constraints (Buchanan et al., 2003; Spencer et al., 2004). In order to determine whether developmental conditions of chicks affect the subsequent development of their song, we manipulated brood size and recorded the songs of first-year reproducing males from experimentally enlarged, reduced, or control broods. Our aim was to test in this complementary experiment whether song characteristics are affected by developmental conditions in blue tits because this hypothesis remains untested so far and is crucial for arguments about adaptive sex ratio adjustment. METHODS The field work was carried out in 2002 and 2003 in the Parc Régional de la Forêt d Orient (Aube, France, 48 17# N, 4 17# E) as part of a long-term study on tit reproduction. Circa 1000 nest-boxes, set in 1998, are evenly spread over a total area of 500 ha. The forest consists mainly of sessile oak (Quercus petraea) and European hornbeam (Carpinus betulus). Nest-boxes were visited every 2 4 days to record the laying date of the first egg. The final clutch size was determined during the incubation period. After 12 days of incubation, the nests were visited every second day to determine hatching date and brood size. Parents were nest trapped 7 14 days after hatching, and the young were ringed 7 10 days after hatching. For all birds, we recorded body mass (to the nearest 0.25 g, using a Pesola balance) and tarsus length (to the nearest 0.1 mm). We collected three to four feathers in the middle of the crown of adult males for color measurements. From 23rd March to 24th April 2002 and 2003, 84 breeding males were observed during the dawn chorus. Observations and recordings began 1 h after astronomical twilight, before blue tit dawn chorus starts, and ended when the female exited the nest-box. All males were recorded during the presumed fertile period of the female, which probably occurs from a few days before the first egg until the day when the penultimate egg is laid (Mace, 1987). We only used male recordings done from 6 days before the first egg until the day of the sixth egg. Song recording and analysis Recordings were made using shotgun microphones Audio Technica AT835B connected to a Marantz PMD101 cassette recorder. Song was analyzed with the computer program Avisoft- SASlab Light ( Blue tit song at dawn is characterized by the repetition of the same strophe during a strophe bout, before the male changes strophe type (Poesel and Kempenaers, 2000; Figure 1). Different features of song output were estimated. Mean is given with SD, and repeatability is given for 10 males, which were recorded twice, using a one-way ANOVA (df ¼ 10,11): (1) mean strophe duration ( ; repeatability: R 2 ¼.85, p ¼.002); (2) singing rate (number of strophes per minute, in strophe bouts where pauses did not exceed 10 s) ( ; repeatability: R 2 ¼.84, p ¼.004); (3) strophe diversity estimated as the number of different strophes divided by the number of strophe bouts ( ; repeatability: R 2 ¼.88, p ¼.002); and (4) mean strophe bout length (total number of strophes per strophe bout) ( ; repeatability: R 2 ¼.89, p ¼.001). As the number of strophes sung during the entire dawn chorus was not significantly repeatable (N ¼ 13, R 2 ¼.66, p ¼.091), we only considered a dawn chorus if it contained more than 69 strophes, which is the upper limit of the 95% confidence interval of strophe bout length. We did not consider drift, which is a measure of performance change over time (Poesel and Kempenaers, 2000), because we did not find any Figure 1 (a) Sonograms of two types of strophes from a male blue tit. The segments represent strophe duration. (b) One example of a sequence of strophes sung at dawn, with the succession of three strophe bouts. Each segment represents a strophe.

3 Dreiss et al. Offspring sex ratio, male song, and male coloration 15 significant repeatability of this measure in our sample (N ¼ 10, R 2 ¼.54, p ¼.35). Color analysis We measured feather reflectance of the crown of 82 males observed at dawn with a portable spectroradiometer (Avantes USB-2000 calibrated from 200 to 850 nm) and a deuteriumhalogen light source (DH-2000 emitting from 215 to 1500 nm) connected with a 1.5-mm-diameter sensor inserted in a miniature black chamber (Théry et al., 2005). Reflectance spectra were taken at 90 relative to a 99% reflectance standard ( nm Spectralon) and to dark current (black velvet background). A reference and dark current calibration were taken before measuring feathers of each individual. For each individual bird, we computed a mean of five reflectance spectra, each measured with two feathers superimposed. Three objective parameters of color perception were calculated according to the method described by Endler (1990). Mean is given with SD and repeatability is given with a one-way ANOVA (df ¼ 82,362). (1) Brightness (spectral intensity) ( ; repeatability: R 2 ¼.89, p,.0001) was estimated by R , the sum of reflectance from 300 to 700 nm. (2) Hue (spectral location) ( ; repeatability: R 2 ¼.81, p,.0001) was defined as k max, the wavelength of maximum reflectance. (3) We used as measure of chroma (spectral purity), UV-chroma (R /R ) ( ; repeatability: R 2 ¼.81, p,.0001), which has been used in several studies of blue tit color (Andersson et al., 1998; Delhey et al., 2003; Griffith et al., 2003; Sheldon et al., 1999). Brood size manipulation In 2002, we performed a brood size manipulation experiment to test for effects of rearing condition on subsequent song performance by randomly transferring either one or two chicks from 39 broods and adding these to another 39 broods with the same hatching date. Chicks were transferred in heated boxes at the age of 1 3 days. Chicks from enlarged broods were all marked with the same color ring, while chicks from reduced broods were marked with another color. Molecular methods Blood was collected in capillary tubes (20 ll) and transferred into tubes containing 150 ll phosphate-buffered saline buffer with 2 mm ethylenediaminetetraacetic acid. Tissues were collected from dead embryos and stored in seven volumes of ethanol 70%. Samples were stored at 6 C, then digested all night at 55 C by adding 3 ll proteinase K 20 mg/ml and 3 ll Tris-NaCl (final concentrations [Tris] ¼ 10 mm; [NaCl] ¼ 25 mm). For DNA extraction, we used the QIAquick96 Kit (Qiagen, Courtaboeuf, France), according to the manufacturer s protocol. Sexing technique The sex of all nestlings from 84 broods was determined by polymerase chain reaction (PCR) amplification of the CHD1- W and CHD1-Z genes with the P2 and P8 primers (Griffiths et al., 1998). PCR products, separated by electrophoresis on 1% agarose gels containing ethidium bromide, were visualized under UV light. As a control, parents were also sexed and were all found to be of the expected sex. The eggs containing no visible embryos were discarded. However, because some of these eggs may still have been fertilized, our measure of the brood sex ratio may not correspond exactly to the primary sex ratio, sensu stricto. Paternity analysis Paternity was analyzed by PCR amplification of five microsatellite loci. The five pairs of primers used are from Dawson et al. (2000) and Tanner et al. (GenBank accession number AF041466). The primers were redesigned for multiplex purposes (detailed information can be obtained from the corresponding author on request). PCR products were separated and analyzed with an automatic monocapillary genetic analyzer Abi310. Due to high polymorphism, the probability of detection of extrapair young was.99. Statistical analyses Analyses were performed using SAS (1999). Sample values are given as mean 6 SE. Only strophe bout length differed significantly from normality in a Kolmogorov-Smirnov test. In order to normalize this variable, we transformed it as ÿlog(1/x). Sex ratio, expressed as the proportion of males in a brood, was analyzed with generalized linear models with binomial error distribution, according to Wilson and Hardy (2002). We performed a simplification of the model by backward elimination of the nonsignificant interactions and variables. Second-degree interactions were included in the model, except interactions between correlated song variables. The significance of the final model was determined by a chi-square test between initial model deviance and final model deviance. Partial broods (never more than two chicks missing) were also included in the analyses as suggested by Fiala (1980). A few missing variables for some of the birds explain slightly varying sample size among tests. The analysis of the difference between extrapair and withinpair sex ratios was also performed with a generalized linear model with binomial error distribution. For each nest containing both extrapair and within-pair chicks, we determined the two measures of sex ratios (extrapair or within-pair). The sex ratio obtained was set as the dependent variable and origin (extrapair or within-pair) as the independent variable. Principal component analysis was used to summarize information of song features with a few independent variables (Table 1). The two first principal components Ch1 and Ch2, explaining 64% of the variance, were retained for further analysis. Only these two first principal components have an eigenvalue greater than 1. Ch1 is mainly explained by variables strophe bout length, strophe duration, and song rate and can be interpreted as song performance. Ch2 is mainly explained by strophe diversity and can be interpreted as song diversity. We analyzed the link between all male characteristics with Pearson correlations. The effect of brood manipulation on male song was analyzed with ANOVA procedures. We used the sequential Bonferroni correction to assess the table-wide type I error rate for multiple tests (Holm, 1979). Chick body mass after manipulation was analyzed with a MIXED procedure, which accounts for the nest as a random effect. Table 1 Loadings of the song variables on the four principal components extracted and cumulative variance associated with each Ch1 Ch2 Ch3 Ch4 Strophe bout length ÿ ÿ0.69 ÿ0.35 Song rate ÿ Strophe duration ÿ Diversity ÿ0.18 Cumulative variance 38% 64% 86% 100%

4 16 Behavioral Ecology RESULTS Brood size manipulation, offspring condition, and song Brood manipulation had an effect on chick condition as reflected by chick body mass (ÿ2, ÿ1, 0, 1, or 2 chicks added) (F 2,1061 ¼ 2.27, p,.023) after controlling for age at measurement (F 2,1061 ¼ 22.83, p,.0001). As expected, chicks from reduced broods were heavier than those from enlarged broods (mean 6 SE for broods reduced by two nestlings: , N ¼ 13; mean 6 SE for broods increased by two nestlings: , N ¼ 17). Five reproducing males were recorded in 2003 from more than 380 chicks from enlarged broods in 2002, while nine males were recorded from the 300 chicks from reduced broods. Song analyses of these males showed no significant differences between the two groups of males for strophe duration, song rate, and strophe diversity (strophe duration: R 2 ¼.04, F 1,11 ¼ 0.47, p ¼.51; song rate: R 2 ¼.09, F 1,11 ¼ 1.26, p ¼.28; strophe diversity: R 2 ¼.01, F 1,11 ¼ 0.06, p ¼.81). However, males that were reared in enlarged broods produced significantly shorter strophe bouts than males reared in reduced broods (Figure 2; R 2 ¼.41, F 1,11 ¼ 7.07, p ¼.024. This relation was no more significant after Bonferroni adjustment for four statistical tests: critical p-value:.05/4 ¼.0125). Male song and color Song rate was correlated with strophe duration and strophe bout length after Bonferroni correction (threshold value: a ¼.008 after correction; Table 2). No significant correlation was found between song features, male crown color characteristics, and male biometrics. Population sex ratio Mean brood sex ratio from the 84 broods was (mean 6 SE, minimum ¼ 0.1, maximum ¼ 1.0). Although sex ratio was strongly biased in some broods, its distribution did not differ significantly from a binomial distribution (v 2 ¼ 1.73, df ¼ 2, p ¼.41). Table 2 Pearson correlation coefficients among male dawn song traits Song rate Diversity Strophe bout length Strophe duration ÿ0.31** 0.14 Song rate * Diversity ÿ0.02 *p,.05; **p,.01 after Bonferroni adjustment for six statistical tests (critical p value:.05/6 ¼.0083;.01/6 ¼.0017). N ¼ 105. There was no significant correlation between laying date and sex ratio bias in the brood (difference from 0.5 brood sex ratio) (linear regression: R 2 ¼.0001, F 1,81 ¼ 0.01, p ¼.75). There was no significant relationship between brood sex ratio bias and clutch size (linear regression: R 2 ¼.0064, F 1,81 ¼ 0.52, p ¼.47). We did not find a significant relationship between sex ratio bias and the number of unhatched eggs (linear regression: R 2 ¼.0011, F 1,81 ¼ 0.08, p ¼.93). Song, color, and brood sex ratio In order to determine whether sex ratio varied with song, color, or morphology, we performed a generalized linear model, with sex ratio as the dependent variable and song and color variables, parental biometrics, laying date, and year as the independent variables. The final model explaining sex ratio in our population contains only the strophe bout length during the dawn chorus (Figure 3; v 2 ¼ 5.68, p ¼.017). Hence, we found no significant relationship between sex ratio and year, laying date, parental biometrics, crown color, or any of the first three song variables (strophe duration, song rate, strophe diversity) in our model. Within-pair sex ratio showed the same tendency, and the final model for within-pair sex ratio was even more significant (v 2 ¼ 6.76, p ¼.009). The final model (strophe bout length) and the initial model (all song variables, color variables, year, and the twoway interactions) did not differ significantly in change of variance (delta deviance ¼ 10.8, df ¼ 11, p ¼.46), showing that no other variable improved the final model. Figure 2 Strophe bout length at dawn of 1-year-old males from experimentally manipulated broods (normalized as ÿlog(1/x)), in relation to the manipulation undergone. The manipulation variable has three values: ÿ1 for decreased brood, 1 for increased brood, and 0 for nonexperimental brood with no manipulation. The males from nonmanipulated broods are a random selection of eight 1-year-old males in the population. Figure 3 Natural brood sex ratio in relation to the mean length of strophe bouts produced by the social male at dawn during the fertile period of the female (transformed as ÿlog(1/x)).

5 Dreiss et al. Offspring sex ratio, male song, and male coloration 17 The model performed with the principal components Ch1 and Ch2 gave a similar result. The final model contained the only variable Ch1 song performance (total sex ratio: v 2 ¼ 4.08, p ¼.043; within-pair sex ratio: v 2 ¼ 4.30, p ¼.038). Extrapair and within-pair sex ratio A total of 49% of the broods contained extrapair chicks. From a total of 850 chicks, 117 were extrapair young. There was no significant difference in sex ratio between within-pair and extrapair chicks (F ¼ 0.21, df ¼ 1, p ¼.65; sex ratio of withinpair offspring: ; sex ratio of extrapair offspring: ). DISCUSSION Strophe bout length (or another variable correlated with strophe bout length) was differentially affected by experimentally modifying rearing conditions in the nest, and observed strophe bout length of the attending male at a nest-box predicted offspring sex ratio. None of the other dawn song and crown color characters accounted for a significant amount of variance, raising the question why strophe bout length may be of more importance as a signal. The length of the strophe bout is supposed to reflect male quality as a short strophe bout is an indicator of neuromuscular tiredness (Carew, 2000). A male might be able to keep singing at a high rate by changing strophe types because each type requires a different use of the song apparatus (Lambrechts and Dhondt, 1988). Thus a male that produces long strophe bouts and that is capable of singing the same strophe a great number of times would reveal his endurance and his ability to perform without fatigue. Analyses of songs produced by the few males from sizemanipulated broods revealed that males from enlarged broods had shorter strophe bouts than males from reduced brood. Although the relation is not significant after Bonferroni correction, the variance explained is rather high (R 2. 40%). This experimental result suggests that nestling development may be linked to acquisition of song, especially strophe bout length. Brood size manipulation affected chick condition, which later may have consequences for access to resources. Consistent with this prediction, Nowicki et al. (2000) found a relationship between biometric measures of nestling great reed warblers (Acrocephalus arundinaceus) and their repertoire size during the first breeding season: well-fed young with a superior size displayed a more diversified song repertoire. Thus, male blue tits may signal their general condition and their ability to respond to developmental stress by producing long sequences of strophes during the dawn chorus. The observed link between brood sex ratio and male strophe bout length might suggest that females use this song characteristic as a cue reflecting the quality of their mate. A male-biased sex ratio in the brood was associated with longer strophe bouts during the dawn chorus. If sons inherit this trait, it may be advantageous for the female to adjust brood sex ratio toward males as sons would be of better quality or would enjoy increased mating success compared to sons produced by males with short strophe bouts (Ellegren et al., 1996; Sheldon et al., 1999). Although we have not tested whether females are able to detect differences in male strophe bout length, this trait has the largest variance among the song variables studied. Females thus have a large range of male phenotypes among which they could choose. Nevertheless, as this link is observational, other hypotheses could potentially explain the relation between brood sex ratio and male dawn chorus. Female blue tits may use other cues of male display than strophe bout length to assess mate quality, and such traits may be indirectly linked to this song variable. Environmental quality might also influence brood sex ratio. A territory with more food for offspring would improve their condition. Thus, parents would increase their fitness by selectively producing sons in a prime quality territory. For example, Appleby et al. (1997) found that the sex ratio of tawny owls (Strix aluco) was related to vole density in the territory. Territorial defense by male tits depends on song output (Krebs et al., 1978), and male singing performance may thus influence territory quality. Moreover, males producing long strophe bouts may be in better overall condition, allowing them to provide their mates with better or more food during the laying period and potentially also provide better or more food for their offspring. In this situation, females should bias their brood sex ratio toward sons because such chicks would be in better general condition. We did not find any influence of male UV plumage on sex ratio, contrary to studies by Griffith et al. (2003) and Sheldon et al. (1999). This may be due to the measurement methods as in these previous studies crown reflectance was determined on live birds while we measured reflectance on feathers. Moreover, Sheldon et al. (1999) measured the plumage during nest building when feathers may be less worn. Plumage may indeed deteriorate and fade during the reproductive period (Örnborg et al., 2002). In contradiction to this hypothetical explanation, Griffith et al. (2003) measured color during nest building and also during chick feeding and obtained similar results. Griffith et al. (2003) and Sheldon et al. (1999) focused on the same Swedish population of blue tits. Hence, the difference in the role of crown color may result from different selection pressures, for example, due to differences in environmental light conditions or due to cultural differences in use of traits among populations. Furthermore, we did not find any correlation between song and crown color features. Therefore, male song and color would represent two aspects of phenotypic quality and convey different types of information on male characteristics. Plumage color might be linked to male condition during molt that takes place several months before reproduction. We did not detect any significant difference in sex ratio between extrapair and within-pair young. This result is consistent with observations from other passerine studies (Saino et al., 1999; Sheldon and Ellegren, 1996; Westerdahl et al., 1997), although the power of these tests is low if the true effect size is small or intermediate. Sheldon and Ellegren (1996) suggested that females are unable to affect the sex ratio of specific eggs. However, if the physiological mechanism leading to sex ratio bias is linked to stimulation of the female by the song of her partner at dawn or to courtship feeding, any control by extrapair males must be limited. Because rearing conditions have a major influence on adult size and strophe bout length, male mates may be of much greater importance in influencing brood sex ratios than extrapair males. Although we found a relationship between strophe bout length and brood sex ratio, overall sex ratio in our population did not differ from binomial expectations. This apparent contradiction may suggest that females do not manipulate the sex ratio to a large extent. Although the mechanism remains unknown, the sex of chicks may be controlled by segregation of sex chromosomes (Ellegren et al., 1996), selective sorting of ova by the female (Emlen, 1997), or selective elimination of embryos of one sex. For the two last hypotheses, the costs in terms of invested resources, or delay in laying date, are obvious. However, we did not find any delay in laying date for broods with extreme sex ratios. If the female selectively eliminates embryos of one sex, there should be fewer eggs in more

6 18 Behavioral Ecology biased broods. However, that was not the case as we did not find a link between brood sex ratio bias and the number of unhatched eggs. This does not support the two latter mechanisms. Consequently, we concur with Charnov (1982) that as the sex ratio may depend on many complex factors, the imprecise estimation of different variables affecting fitness would favor small biases and few extreme brood sex ratios. Our data suggest a link between a component of male song and brood sex ratio in the blue tit. Previous studies of the same species have shown relationships between brood sex ratio and parental characteristics. Male survival may play a role in determining sex ratios (Griffith et al., 2003; Svensson and Nilsson, 1996; but see Leech et al., 2001), as may clutch size and breeding time (Griffith et al., 2003; but see Leech et al., 2001). These relationships are not always consistent in the same population in different years (Griffith et al., 2003), although consistency may occur within couples (Oddie and Reim, 2002). This suggests that it is important to determine the relative importance of these factors in determining brood sex ratio and the selection pressures that lead to such patterns. We thank the students for assistance with field work, especially Claire Loiseau, Lorien Pichegru, Nadia Silva, and Izabella Dworzynska. We also gratefully acknowledge Alain Jamet for his technical assistance. We would like to thank the French national forestry agency Office National des Forêts and the administration of the Parc Régional de la Forêt d Orient for their kind collaboration. The work was partly funded by the GDR2155 d Ecologie Comportementale. 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