Extra-pair paternity, offspring mortality and offspring sex ratio in the socially monogamous coal tit (Parus ater)

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1 Behav Ecol Sociobiol (2006) 60: DOI /s ORIGINAL ARTICLE Verena Dietrich-Bischoff. Tim Schmoll. Wolfgang Winkel. Sven Krackow. Thomas Lubjuhn Extra-pair paternity, offspring mortality and offspring sex ratio in the socially monogamous coal tit (Parus ater) Received: 13 September 2005 / Revised: 6 March 2006 / Accepted: 23 March 2006 / Published online: 20 June 2006 # Springer-Verlag 2006 Abstract Females of many socially monogamous bird species commonly engage in extra-pair copulations. Assuming that extra-pair males are more attractive than the females social partners and that attractiveness has a heritable component, sex allocation theory predicts facultative overproduction of sons among extra-pair offspring (EPO) as sons benefit more than daughters from inheriting their father s attractiveness traits. Here, we present a largescale, three-year study on sex ratio variation in a passerine bird, the coal tit (Parus ater). Molecular sexing in combination with paternity analysis revealed no evidence for a male-bias in EPO sex ratios compared to their withinpair maternal half-siblings. Our main conclusion, therefore, is that facultative sex allocation to EPO is absent in the coal tit, in accordance with findings in several other species. Either there is no net selection for a deviation from random sex ratio variation (e.g. because extra-pair mating may serve goals different from striving for attractiveness Communicated by S. Pruett-Jones V. Dietrich-Bischoff Zoological Institute, TU Braunschweig, Spielmannstraβe 7, Braunschweig, Germany T. Schmoll. T. Lubjuhn (*) Institute for Evolutionary Biology and Ecology, University of Bonn, An der Immenburg 1, Bonn, Germany t.lubjuhn@uni-bonn.de Tel.: Fax: W. Winkel Institute of Avian Research Vogelwarte Helgoland, Working Group Population Ecology, Bauernstraße 14, Cremlingen, Germany S. Krackow Institute for Biology, Humboldt-University of Berlin, Invalidenstraße 43, Berlin, Germany genes ) or evolutionary constraints preclude the evolution of precise maternal sex ratio adjustment. It is interesting to note that, however, we found broods without EPO as well as broods without mortality to be relatively female-biased compared to broods with EPO and mortality, respectively. We were unable to identify any environmental or parental variable to co-vary with brood sex ratios. There was no significant repeatability of sex ratios in consecutive broods of individual females that would hint at some idiosyncratic maternal sex ratio adjustment. Further research is needed to resolve the biological significance of the correlation between brood sex ratios and extra-pair paternity and mortality incidence, respectively. Keywords Coal tit. Parus ater. Extra-pair paternity. Sex allocation. Brood sex ratio. Offspring mortality. Molecular sexing Introduction Extra-pair paternity (EPP) has proven to be common among socially monogamous bird species (reviewed by Birkhead and Møller 1992; Griffith et al. 2002). Females regularly engage in extra-pair copulations (EPCs) with males other than their social mate which leads to offspring genetically unrelated to the social father providing care (extra-pair offspring, EPO). Concerning potential benefits of EPCs to females, it is generally assumed that female birds express a mating preference for males of higher genetic quality compared to their social mate and, thus, obtain genetic benefits for their offspring (in terms of viability and/or attractiveness genes, e.g. Jennions and Petrie 2000; Griffith et al. 2002; Kokko et al. 2002). Such genetic benefit models of female (extra-pair) mate choice can be combined with sex allocation theory (Trivers and Willard 1973; Charnov 1982) which predicts that parents should be selected to overproduce the more valuable sex whenever sons and daughters yield different fitness benefits and/or entail different costs (reviewed in Clutton-Brock 1986; Hardy 1997; Cockburn et al. 2002).

2 564 Especially in polygynous (e.g. Hartley et al. 1999) but also in socially monogamous species with EPP (e.g. Møller and Ninni 1998), the potential rate of reproduction as well as the variance in fertilisation success is greater for males compared to females. The reproductive value of sons would, thus, be likely to exceed that of daughters if sons were especially attractive to females, promising potentially high (extra-pair) mating success. Accordingly, one prediction derived from sex allocation theory is that females should bias their broods towards sons when mated to attractive males (e.g. Burley 1981, but see Pen and Weissing 2000 for critical discussion), assuming heritable genetic variation for male attractiveness and that sons benefit more than daughters from inheriting their father s attractiveness traits. If females actually engaged in EPCs with more attractive males, they should, therefore, benefit from biasing the sex ratio of EPO (but not of within-pair offspring; WPO) towards sons. Alternatively, broods with EPO should be male-biased in case females do not possess sufficient physiological control to manipulate the sex of an individual egg with respect to paternity (e.g. Sheldon and Ellegren 1996). Indeed, an early study in the blue tit (Parus caeruleus) found EPO sex ratios to be male-biased (Kempenaers et al. 1997), suggesting adaptive maternal sex ratio adjustment. However, this finding, based on a morphological determination of nestling sex, could not be corroborated in a number of studies in different (Westneat et al. 1995; Sheldon and Ellegren 1996; Westerdahl et al. 1997; Saino et al. 1999; Cordero et al. 2000; Questiau et al. 2000; Magrath et al. 2002; Ramsay et al. 2003) or even the same species (Leech et al. 2001). The main aim of this study is to evaluate the validity of sex ratio differences between EPO and WPO and between broods with and without EPP in an established nestbox population of a socially monogamous passerine bird, the coal tit (P. ater). The consistently high rate of EPP in this population (Dietrich et al. 2004) clearly has the potential to increase the variance in male realised (i.e. genetic) reproductive success as there is usually no reciprocal exchange of extra-pair paternities between territorial males (own unpublished data). The selection pressure on females to overproduce sons when mated to an attractive (extrapair) male might actually be higher in coal tits compared to other (tit) species with moderate rates of EPP (e.g. blue tits; cf. Appendix in Griffith et al. 2002) and easier overcome potential constraints imposed on sex ratio adjustment (e.g. Williams 1979). A sex ratio bias as predicted should, therefore, be more easily detectable in such species. Additionally, we checked for potentially confounding influences of variables that might reflect parental or environmental quality and were shown to affect offspring sex ratio in a number of other bird species (i.e. phenotypic traits of the social parents, clutch size and breeding date; for review, see Bensch 1999; Komdeur and Pen 2002). We also contrasted broods with and without mortality until blood sampling as the possibly foremost indicator of current investment conditions. Finally, we tested whether brood sex ratios were repeatable for individual females in consecutive breeding attempts. Materials and methods Study population and field methods We studied an established nestbox population of coal tits in a mixed coniferous forest near Lingen/Emsland (Lower Saxony, Germany, N, 7 15 E) over 3 years ( , additionally, some recapture data from 2003 were included). Coal tits are small (about 10 g) territorial, altricial hole-breeding passerine birds with biparental care (Glutz von Blotzheim and Bauer 1993). Although they are socially monogamous (Glutz von Blotzheim and Bauer 1993) with social pair bonds often being maintained within as well as between years (Winkel and Winkel 1980), EPP has been shown to be common in this species (Lubjuhn et al. 1999; Dietrich et al. 2004). In the study population, 70.8% of broods and 31.4% of nestlings were, on average, affected by EPP over 3 years (Dietrich et al. 2004). These rates surpass all those revealed in other species of the wellinvestigated genus Parus (titmice and chickadees) by far (cf. Appendix in Griffith et al. 2002). The 325 ha study area contained about 560 nestboxes (details in Winkel 1975) in which coal tits regularly breed in high numbers (2000: 132 pairs, 2001: 184 pairs, 2002: 177 pairs). The proportion of second broods in the population may vary between 0 100% in different years (Winkel and Winkel 1997); in 2000, it amounted to 63.5%, in 2001, to 1.2% and in 2002, to 31.3%. Broods were considered second broods when they followed a successful first brood. A few broods could not unequivocally be identified as second broods but because the first and second brood period did not overlap in any year, these broods were classified according to their hatching date for statistical analyses. During the breeding seasons (April to July), nestboxes in the study area were checked at least weekly. Adults were captured whilst feeding nestlings days old and regarded the social parents of the respective brood. At the same time, both adults (if unbanded) and nestlings were banded with unique numbered rings of the Vogelwarte Helgoland and blood samples (about 50 μl) were taken from the ulnar vein. There was no evidence that blood sampling affected fledging success or local recruitment probability into the study population (Schmoll et al. 2004). Breeding adults were sexed by presence or absence of a brood patch. They could be classified young (1 year old) or old (2 years or older) according to plumage characteristics (see Jenni and Winkler 1994). Furthermore, about twothirds of breeding adults had been banded as nestlings and their exact age was therefore known. Body mass of adults was taken to a precision of 0.1 g using an electronic balance. In 2001 and 2002, tarsus length and wing length of adults were measured with a calliper to a precision of 0.1 and 0.5 mm, respectively. Size and colour saturation of the sexually dimorphic blackish throat feathering ( bib ) were determined according to the scales suggested by King and

3 565 Griffiths (1994) who proposed five size and three saturation classes with intermediate (0.5) intervals. Survival to the next breeding season was inferred from recapture data because, owing to the distinct breeding site fidelity in the coal tit (e.g. Winkel and Winkel 1980), recapture rates serve as a good measure for actual survival rates. However, data were inconsistent in a few cases where adult birds were not recorded in the next breeding season but in a later one. For two reasons, we decided to use data on recapture in the following year only. Firstly, merely 1.8% of 275 females and 3.9% of 279 males were concerned. And, secondly, the probability to correct an incorrect survival estimate was higher for breeders first recorded in 2000 compared to those sampled in a later year. We recorded several parameters of reproductive performance, i.e. clutch size, hatching date and number of nestlings. Hatching was either observed directly or hatching date was inferred from the age of young nestlings (see Winkel 1970) during the next nestbox inspection. The presence of unhatched eggs and nestlings that died before blood sampling was recorded as well. Parentage analysis The results reported here are based on multilocus DNA fingerprinting analyses. Because all details on the procedures have been described previously (Epplen 1992; Lubjuhn and Sauer 1999), the methods are only briefly summarised. Blood samples were diluted in 250 μl APS buffer (Arctander 1988) and stored at 20 C until further analyses. DNA was isolated according to a modified standard procedure (Lubjuhn and Sauer 1999) and digested with the restriction enzyme Hae III following the instructions given by the manufacturer. The digests were separated by horizontal gel electrophoresis (gel size cm, 0.8% agarose, 1 2 V/cm) in 1 TBE buffer. Gels were then dried followed by in-gel-hybridisation using the 32 P labelled oligonucleotide (CA) 8. The resulting banding patterns were analysed according to the rules outlined by Westneat (1990). The data obtained allowed parentage analyses in the coal tit as demonstrated elsewhere in detail (Lubjuhn et al. 1999) with the probability of falsely assigning one putative parent to an offspring being as low as (Dietrich 2001). Molecular sex determination Only hatchlings that survived until blood sampling (between day 10 and 14) were sexed (with the exception of nine nestlings in 2001 and two nestlings in 2002 that died shortly before blood sampling and were not thrown out of the nestbox by their parents; they could, thus, be sexed on the basis of tissue samples). For molecular determination of nestling sex we used the method developed by King and Griffiths (1994) based on a primer pair (RG 11 and RG 12, for sequence information see King and Griffiths 1994) which amplifies a W-chromosomal, i.e. female-specific locus in the coal tit via polymerase chain reaction (PCR). To test the individual DNA samples we added another primer pair (2 KM-5 and 2 KM-3) as a positive control that was originally developed for microsatellite analyses in the great tit but amplifies an anonymous locus in coal tits as well (Gerken 2001). After PCR, agarose gel electrophoresis (gel size 7 10 cm, 2% agarose, 130 V/cm) and staining with ethidium bromide, this procedure yields only one visible fragment for males and two distinct fragments for females. PCR conditions were as follows: Approximately 100 ng of genomic DNA were amplified in the presence of the primers 2 KM-5 and 2 KM-3 (20 pmol each) and the primers RG 11 and RG 12 (5 pmol each). Each PCR reaction additionally contained 200 μm dntps, 4 mm MgCl and 0.25 U Taq polymerase. Cycling conditions included initial denaturing for 4 min at 94 C, followed by 6 cycles with 30 s at 94 C, 1 min at 66( 1) C, and 40 s at 72 C, and then 28 cycles with 10 s at 94 C, 20 s at 60 C and 15 s at 72 C. Final extension lasted for 10 min at 72 C. Statistical analysis Analyses were carried out using SAS programming and software (SAS Institute Inc. 1989). Overall sex ratio bias (deviation from 50% males) was evaluated using an optimal test that takes into account broods as independent data points by adjusting statistics using the within-brood correlation of sexes (Neuhäuser 2004). Deviations of sex ratio variance as well as variance of offspring mortality from binomial expectation were investigated using James procedure for overdispersion (Krackow et al. 2002). Both methods yield standard normal deviate (z) test statistics. The sex ratios of EPO and WPO within broods were compared using conditional logistic analysis which accommodates for the potential dependency of sexes within broods by taking brood identities for representing the strata and yields a χ 2 test statistic (Molenberghs 2002; Neuhäuser, personal communication). Differences between brood sex ratios (proportion males) of different groups and/or in response to covariates were analysed with generalised linear models (McCullagh and Nelder 1989), assuming binomial error distribution (with number of males as nominator and brood size as denominator) and taking logits as the response. In these models, variance was scaled by Pearson s chi-square for G.O.F. which concomitantly yielded likelihood-ratio based F-tests for significance of parameters, i.e. broods rather than individual chicks were taken as independent data points (Krackow and Tkadlec 2001). In multiple effects models, each term was corrected for all other effects in the analyses (type 3 or simultaneous models). For multiple explorative analyses of parental trait effects, all independent variables were initially included in a simultaneous model. The least significant effect was then removed, yielding a reduced model that was treated the same way until no or only significant effects remained.

4 566 To avoid pseudo-replication due to parents contributing more than one brood to the sample, generalised linear mixed models proved unreliable, mostly producing random covariance estimates of 0. This might have been caused by 155 of 275 females (56.4%) producing only a single brood and the random component lying very close to binomial expectation. Hence, as a conservative measure to avoid pseudo-replication, we used the F-statistics from fixed effect models and, for significant effects, calculated error degrees of freedom as the number of mothers contributing broods minus model degrees of freedom. To test whether brood sex ratios of individual females were repeatable, we compared consecutive broods by repeatability analysis (Lessells and Boag 1987). Other standard statistical tests were used and are identified with the results. Two-sided error probabilities are given throughout and the null hypothesis was rejected at P<0.05. Sample sizes may vary between different analyses when not all measures could be taken for all broods. Results The entire data set comprised 3,492 coal tit nestlings sexed in 471 first and second broods produced by 275 different females over 3 consecutive years (Table 1). Sexed chicks constituted 90.9% of eggs laid (n=3,843) and 95.7% of hatchlings (n=3,647). The overall sex ratio (49.1% males) was close to equality (z=1.05, P=0.29). Brood sex ratio variance appeared to be close to binomial expectation (z=1.48, P=0.13), exhibiting negligible negative withinbrood correlation of the sexes (ρ= 0.01). Hence, there was no indication of any significant overall sex ratio bias or sex ratio variation in the study population. Effects of paternity and offspring mortality on brood sex ratios 457 of the 471 broods sampled for molecular sexing could be analysed with regard to paternity as well. 1,057 of the 3,365 nestlings genotyped within these broods (=31.4%) resulted from extra-pair fertilisations (EPFs, Table 1). In 23 broods, only EPO occured, 299 multiply sired broods contained EPO as well as WPO and no EPO were found in 135 broods. Of the latter, 67 were completely genotyped (i.e. all eggs hatched and all hatchlings survived until blood sampling) so that EPFs could definitely be ruled out in these cases. Comparing the sex ratios of EPO and WPO within multiply sired broods by conditional logistic analysis suggested no difference between these two groups of offspring (χ 2 1=0.88, P=0.35, Table 1), i.e. mothers did not discriminate between EPO and WPO with respect to sex determination. Moreover, there was no correlation between EPO and WPO sex ratios within broods as shown by nonsignificant Spearman s rank correlation (R s =0.007, P=0.91). However, generalised linear analysis revealed a significant difference of sex ratios between broods containing EPO and those that did not (F 1,273 =3.96, P=0.048, Table 1). Whilst the sex ratio of broods with EPP was balanced (50.0% males), broods without EPP were biased towards females (46.3% males). This finding remained stable when broods without EPP that could not be completely genotyped (i.e. could possibly have contained EPO) were excluded (F 1,240 =5.28, P=0.03). Within 136 of the 469 broods with known clutch size (29.0%), we found 201 unhatched eggs. Assuming an independent individual egg mortality risk, 35.4% of broods would have been expected to be affected, given the 5.2% egg mortality recorded. Consequently, egg mortality was strongly overdispersed (z=8.60, P<0.001). Those broods not affected by egg mortality were significantly femalebiased compared to affected broods (F 1,467 =5.00, P=0.03). Simultaneous two-factor analysis showed independent effects of the occurrence of EPP (F 1,265 =4.52, P=0.04) and the incidence of egg mortality within a given brood (F 1,265 =5.91, P=0.02) on brood sex ratio. A saturated model revealed no significant interaction either (F 1,264 =0.005, P=0.98). Hence, females produced relatively more daughters when their broods did not contain EPO or when their broods did not suffer from egg mortality, and these effects appeared additive (Fig. 1). Table 1 Data set used for analysis of sex ratio variation in first and second broods of coal tits from 3 consecutive years Year Brood period Broods sexed Hatched Males Females Broods genotyped No EPP M/F WPO M/F EPO M/F / / / /35 94/72 90/ / / / /5 2/3 1/ / / / /14 52/54 55/72 Total 471 3,647 1,715 1, / / /537 Columns indicate year, brood period (first or second brood), number of broods analysed as well as number of nestlings hatched and sexed in these broods. Concerning broods genotyped with respect to paternity, number of males (M) and females (F) are given for broods without extra-pair offspring (No EPP) and for within-pair (WPO) and extra-pair offspring (EPO) in broods with EPP. Because not all hatchlings could be sexed and not all chicks sexed could be genotyped for paternity analysis, there are fewer chicks sexed than hatched and fewer offspring genotyped than sexed

5 567 effect on brood sex ratios in the simultaneous generalised linear model (Table 2). Stepwise variable exclusion (see Materials and methods section) left no significant effect. Clutch size at laying as well as brood size at hatching, potentially reflecting immediate parental investment conditions, did not affect brood sex ratios either (clutch size F 1,467 =0.04, P=0.83, brood size F 1,469 =0.25, P=0.61). In contrast to brood sex ratios, the incidence of EPP and mortality, respectively, was related to some parental traits. The probability of EPP increased with wing length of the social father (F 1,218 =4.27, P=0.04). Furthermore, there was a positive correlation between the occurrence of mortality and maternal body mass (F 1,217 =5.61, P=0.02) as well as maternal bib colour saturation (F 1,217 =8.64, P=0.004). Between- and within-year variability of brood sex ratios, EPP and offspring mortality Fig. 1 Brood sex ratios (estimated proportion of males per brood ±95% confidence interval) in relation to extra-pair paternity (EPP; broods with vs without extra-pair offspring, EPO) and mortality (broods affected vs not affected by egg mortality; see Materials and methods section) The occurrence of EPP and egg mortality were not significantly associated (Fisher s exact P=0.17). These differences in sex ratios between broods with and without EPP on the one hand and broods with and without egg mortality on the other hand are expected to increase the variance in brood sex ratios. However, overall sex ratio variance did not significantly depart from random variation (see above). This implies that in some sub-sample(s), sex ratio variance must have been reduced to compensate for the variance increasing effect of the above differences (cf. Krackow et al. 2002). Accordingly, we found that sex ratio variance was significantly reduced in broods without EPP and without egg mortality (z=2.02, P=0.049, ρ= 0.02) but not in those without EPP exhibiting egg mortality or in those with EPP, with and without egg mortality (z<1.1, P>0.27 in all cases). All above-mentioned statistical analyses which concerned the occurrence of egg mortality within broods yielded the same results when dead nestlings, or both unhatched eggs and dead nestlings, were used instead of unhatched eggs only (data not shown). Effects of parental traits on brood sex ratios, EPP and offspring mortality Because the sex ratio difference between broods with and without EPP on the one hand and broods with and without mortality on the other hand might actually originate from another factor, we tested the effect of several parental and environmental variables on brood sex ratios, EPP and mortality. Neither maternal and paternal age, survival, body mass, tarsus length, wing length nor bib size and bib colour saturation (see Materials and methods section) had an Neither the year of study, brood period nor their interaction had detectable effects on brood sex ratios (year F 2,465 =2.12, P=0.11, brood period F 1,465 =0.36, P=0.54, year brood period F 2,465 =2.01, P=0.13). Within first and second brood periods, hatching date did not influence sex ratios significantly (first brood period F 1,363 =0.37, P=0.54, second brood period F 1,104 =0.64, P=0.42). These statistical inferences also held when EPO were excluded from analyses as well as for EPO only (P>0.1 in all cases). Hence, no shifts in brood sex ratios between different years or first and second broods within years became obvious, either for WPO or for EPO. Concerning the occurrence of mortality and EPP, respectively, the incidence of mortality did not vary significantly between years and brood periods (year F 2,465 =1.74, P=0.17; brood period F 1,465 =1.88, P=0.17) whilst significantly more broods contained EPO in second than in first brood periods (year F 2,453 =0.10, P=0.90; brood Table 2 Effects of the social parents traits on offspring sex ratios in a simultaneous generalised linear model including 235 broods from 192 females which provided measures of all independent variables Independent variable F 1,220 P Social mother Age category Survival Body mass Tarsus length Wing length Bib saturation Bib size Social father Age category Survival Body mass Tarsus length Wing length Bib saturation Bib size

6 568 period F 1,453 =10.20, P=0.002; cf. Table 1 and Dietrich et al. 2004). Year by brood period interaction terms were not significant in either case. Within first and second brood periods, neither the incidence of mortality (first brood period F 1,361 =2.25, P=0.13, second brood period F 1,104 =0.19, P=0.66) nor the occurrence of EPP (first brood period F 1,351 =1.51, P=0.22, second brood period F 1,102 =0.04, P=0.85) varied with hatching date. Repeatability of brood sex ratios To analyse patterns of brood sex ratios for individual females, we performed repeatability analyses for those females with more than one brood sampled and sexed. First, we compared offspring sex ratios between first and second broods of the same year. Only females breeding with the same mate were included (because coal tits show very high social mate fidelity within a breeding season, there were only four cases of mate change). The 2 years with an adequate proportion of second broods, 2000 and 2002, were pooled. To avoid pseudo-replication, four females with two broods in both these years were considered only for the 2000 data set. Brood sex ratios proved to be not repeatable between first and second broods (n=78, r= 0.22, F 77,78 =0.64, P=0.97). Furthermore, we analysed 87 females with two consecutive first broods. Females were split in two groups, individuals with retention and change of their social mate. In neither group was there a significant repeatability of brood sex ratios (mate retention n=45, r= 0.15, F 44,45 =1.35, P=0.16; mate change n=42, r= 0.24, F 41,42 =1.64, P=0.06). There were not enough individuals with two consecutive second broods to perform this analysis for second broods. Discussion In a large sample of coal tit broods exhibiting no overall bias in sex ratio, we found no difference between EPO and WPO sex ratios within multiply sired broods. Hence, there seems to be no effect of paternity on individual offspring sex in this species. This finding is consistent with the results from empirical studies in red-winged blackbirds Agelaius phoeniceus (Westneat et al. 1995), collared flycatchers Ficedula albicollis (Sheldon and Ellegren 1996), great reed warblers Acrocephalus arundinaceus (Westerdahl et al. 1997), house sparrows Passer domesticus (Cordero et al. 2000), bluethroats Luscinia svecica (Questiau et al. 2000), fairy martins Petrochelidon ariel (Magrath et al. 2002) and black-capped chickadees Poecile atricapilla (Ramsay et al. 2003) as well as with an experimental study in barn swallows Hirundo rustica (Saino et al. 1999). The only support for an association between paternity and EPO sex ratio comes from a Belgian population of blue tits in which EPO were more likely to be male compared to WPO (Kempenaers et al. 1997). However, in another population of the same species, Leech et al. (2001) found no such difference when using a molecular sexing technique (instead of a method based on morphology) and when relying on a larger data set. All these studies were conducted in bird species with low or moderate rates of EPP whereas the coal tit exhibits comparatively high EPP rates (Dietrich et al. 2004, cf. Appendix in Griffith et al. 2002) which is likely to substantially increase the variance in male fertilisation success. Hence, potential benefits to females of adjusting offspring sex according to paternal attractiveness might be especially pronounced in the coal tit. However, our failure to find the expected relationship may be due to several different reasons. For example, there might be no net selective pressure on females to deviate from random sex ratio allocation as imposed by Mendelian segregation. The assumption of a difference in attractiveness between a female s extra-pair and social male (and, accordingly, between male EPO and WPO) might be wrong and, thus, it would entail no benefit to bias EPO sex ratios towards sons. Likewise, evolutionary constraints, like opposing offspring genetic interests (e.g. Krackow 2002), might prevent the evolution of facultative maternal sex ratio adjustment, or potential benefits of overproducing sons might be diminished by higher production costs of sons compared to daughters. In the study population, nestlings showed a slight sexual size dimorphism with males being larger (own unpublished data) but we were unable to evaluate whether sons were actually more costly for parents to produce. However, this aspect may be of greater relevance in species with a more pronounced sexual size dimorphism (cf. Stauss et al. 2005), like raptors (see e.g. Rosenfield et al. 1996). Finally, manipulating the sex of an individual offspring with respect to paternity would require finely tuned reproductive processes and females might simply not possess sufficient physiological control to determine which male s sperm fertilises which egg. This idea is possibly supported by findings from a Swedish collared flycatcher population in which brood sex ratio was positively related to the size of the social father s forehead patch (Ellegren et al. 1996). Although this heritable secondary sexual character also predicted male extra-pair mating success, EPO were not more likely to be male (Sheldon and Ellegren 1996). Instead of a difference between EPO and WPO sex ratios, we found a sex ratio difference between broods with and without EPP such that broods without EPP contained relatively more daughters. This finding might actually be considered evidence for a lack of physiological control by females they bias their whole brood towards sons because they are incapable of manipulating the sex of individual offspring. However, it is difficult to explain why broods without EPP contained more daughters than expected under random sex ratio variation, instead of broods with EPP (which exhibited a completely even sex ratio) containing more sons. Furthermore, a comparison of broods with and without EPP is problematic, as it remains unknown why part of the broods did not contain EPO. On the one hand, EPCs might well have taken place but have not resulted in EPFs (e.g. Hunter et al. 1992) due to sperm competition and/or cryptic female choice biasing

7 569 fertilisation success towards the social mate. This could be the case if extra-pair mating aimed at fertility insurance or genetic compatibility in this species. On the other hand, females without EPO might not have engaged in EPCs. They might have been willing to do so but been restricted (e.g. through male mate guarding), and should then produce broods with even or female-biased sex ratios. Alternatively, they might not have pursued EPCs because they were mated to attractive social males so that they are expected to bias their broods towards sons, just like females with EPO. As a consequence, it remains unclear whether females without EPO actually have different quality partners than females with EPO, i.e. the attractiveness hypothesis of sex ratio adjustment need not necessarily hold true. The relevance of mating with more attractive males for brood sex ratio manipulation has generally been questioned because the predicted association could not be found in many bird species (e.g. Saino et al. 1999; Radford and Blakey 2000; Grindstaff et al. 2001; Ramsay et al. 2003), even if several other studies reported a positive correlation between the proportion of sons and (potential) indicators of male attractiveness (e.g. Ellegren et al. 1996; Sheldon et al. 1999; Yamaguchi et al. 2004). Furthermore, the findings of two classic studies which supported the above hypothesis by experimentally manipulating male attractiveness in zebra finches Taeniopygia guttata (Burley 1981, 1986) could not be replicated by Rutstein et al. (2005) when controlling for possibly confounding effects like assortative mating. Finally, using an evolutionary stable strategy (ESS) analysis, Pen and Weissing (2000) showed that it may depend on the mechanism of sexual selection underlying the evolution of male attractiveness whether or not females should adjust the sex ratio of their offspring in relation to paternal attractiveness. According to their results, a sex ratio bias would not necessarily be expected under a purely Fisherian runaway process. In the coal tit study population, brood sex ratios were not influenced by paternal traits potentially reflecting attractiveness, i.e. biometric measures and the size and colour saturation of a sexually dimorphic plumage trait, the bib. However, in contrast to other bird species in which the role of such traits in mate choice is well known (e.g. Ellegren et al. 1996), their significance for coal tit mate choice remains to be analysed. The sex ratio difference we found between broods with and without EPP might originate from a factor that is not related to paternal attractiveness but co-varies with the occurrence of EPP, i.e. a factor that differs between females with and without EPO. For instance, EPP might correlate with some environmental or parental quality variable that could select for sex ratio adjustment as well because mothers of high investment capability would be predicted to overproduce the sex that benefits more from additional investment (Trivers and Willard 1973); in this study, males. Accordingly, sex ratio differences between broods with and without EPP might ultimately relate to differences in sexspecific prospects and, thus, we explored the influence of measures of parental and environmental quality. However, neither the occurrence of EPP nor brood sex ratios were significantly related to parental age as a potential indicator of breeding experience (cf. Leech et al. 2001, but see Risch and Brinkhof 2002), survival to the next breeding season (cf. Leech et al. 2001, but see Griffith et al. 2003), and body condition (cf. Saino et al. 1999, but see Whittingham and Dunn 2000) as well as clutch size (cf. Ramsay et al. 2003, but see Dyrcz et al. 2004) which could both reflect some aspect of investment capability. Although the incidence of EPP correlated with a decline in environmental conditions (with significantly higher EPP rates in second compared to first broods, see also Dietrich et al. 2004), there was no seasonal variation in sex ratio. We found no correlation between brood sex ratios and year (cf. Leech et al. 2001, but see Koenig and Dickinson 1996), brood period (cf. Magrath et al. 2002, but see Lessells et al. 1996) and hatching date (cf. Verboven et al. 2002, but see Krebs et al. 2002) whilst the environmental conditions in the study area varied considerably. But despite a general recruitment advantage of hatching earlier (Dietrich et al. 2003; Schmoll et al. 2005) and despite different recruitment probabilities between years (Schmoll et al. 2005), there was no interaction effect of sex and hatching date or year, respectively, on recruitment success in our population (own unpublished data). Thus, a bias in sex ratio with respect to breeding date does not seem to be adaptive to females. However, the foremost indicator of current investment conditions should be the occurrence of mortality within broods. Hence, we contrasted broods with and without mortality (of eggs and nestlings, respectively) and actually found a difference in sex ratio broods spared from mortality were significantly female-biased compared to broods affected by mortality. However, it should be noted that sex ratio differences at the time of blood sampling cannot arise as a consequence of sex-specific mortality because, if differential mortality existed, those broods escaping mortality would be biased in favour of the sex with the higher survival prospects from the start (Fiala 1980). The effects of EPP and mortality were independent and additive so that the sex ratio difference with respect to mortality could not be explained by the sex ratio difference with respect to EPP and vice versa. Furthermore, just like for the incidence of EPP, the difference between broods with and without mortality could not be explained by potentially confounding environmental or parental quality variables. Only maternal body mass and bib colour saturation had an effect on mortality but not on sex ratio mortality occurred significantly more often in broods of heavier females and in broods of females with darker bibs. This seems counterintuitive but might possibly indicate that those females invested less resources in breeding (which could then have led to an increase in offspring mortality). The results reported above would have possibly been confounded if there had been consistent differences in sex ratio variation between individual females, i.e. if some idiosyncratic maternal sex ratio adjustment had existed. However, sex ratios were not repeatable for consecutive broods of individual females (cf. Leech et al. 2001, but see

8 570 Griffith et al. 2003), also when splitting the sample in individuals with mate change and mate retention (but see Oddie and Reim 2002 who reported repeatable sex ratios only for females staying paired with the same mate). In conclusion, EPO sex ratios were not male-biased within multiply sired coal tit broods, in contrast to some theoretical expectations. However, we found a sex ratio difference between broods with and without EPP as well as between broods with and without mortality. These differences were not due to parental and environmental variables co-varying with EPP or mortality and selecting for sex ratio adjustment as well. Furthermore, there were no consistent differences in brood sex ratios between individual females. Only further research can resolve the biological significance of the correlation between brood sex ratios and EPP and mortality incidence, respectively. Acknowledgements We would like to thank Sabrina Bleidissel, Anke Kalt, Maria Orland, Andrea Petzold, Tanja Meißner and Christiane Wallnisch for their help in the laboratory, Jörg Brün, Thomas Gerken, Volker Janzon, Anja Quellmalz, Jorg Welcker and Doris Winkel for assistance in the field, Karin and Herbert Körner for housing and hospitality during field work and Georg Rüppell for the provision of working facilities. We thank three anonymous referees for valuable comments on an earlier draft of the manuscript. The project was financed by the Deutsche Forschungsgemeinschaft (Lu 572/2-4) and VD-B was supported by a Ph.D. scholarship provided by the TU Braunschweig. This research was conducted under licence by the competent German authority (No. 509f ). References Arctander P (1988) Comparative studies of avian DNA by restriction fragment length polymorphism analysis: convenient procedures based on blood samples from live birds. J Ornithol 129: Bensch S (1999) Sex allocation in relation to parental quality. In: Adams NJ, Slotow RH (eds) 22nd International Ornithological Congress, Durban, August BirdLife South Africa, Johannesburg, South Africa, pp Birkhead TR, Møller AP (1992) Sperm competition in birds: evolutionary causes and consequences. Academic Press, London, UK Burley N (1981) Sex ratio manipulation and selection for attractiveness. Science 221: Burley N (1986) Sex-ratio manipulation in color-banded populations of zebra finches. Evolution 40: Charnov EL (1982) The theory of sex allocation. Princeton University Press, Princeton, New Jersey Clutton-Brock TH (1986) Sex ratio variation in birds. Ibis 128: Cockburn A, Legge S, Double MC (2002) Sex ratios in birds and mammals: can the hypotheses be disentangled? In: Hardy ICW (ed) Sex ratios: concepts and research methods. Cambridge University Press, Cambridge, UK, pp Cordero PJ, Griffith SC, Aparicio JM, Parkin DT (2000) Sexual dimorphism in house sparrow eggs. Behav Ecol Sociobiol 48: Dietrich V (2001) Zum Auftreten alternativer Fortpflanzungsstrategien in einer Lingener Population der Tannenmeise Parus ater. Diploma thesis, Technical University of Braunschweig, Germany Dietrich VCJ, Schmoll T, Winkel W, Lubjuhn T (2003) Survival to first breeding is not sex-specific in the coal tit (Parus ater). J Ornithol 144: Dietrich V, Schmoll T, Winkel W, Epplen JT, Lubjuhn T (2004) Pair identity an important factor concerning variation in extra-pair paternity in the coal tit (Parus ater). Behaviour 141: Dyrcz A, Sauer-Gürth H, Tkadlec E, Wink M (2004) Offspring sex ratio variation in relation to brood size and mortality in a promiscuous species: the aquatic warbler Acrocephalus paludicola. Ibis 146: Ellegren H, Gustafsson L, Sheldon BC (1996) Sex ratio adjustment in relation to paternal attractiveness in a wild bird population. Proc Natl Acad Sci USA 93: Epplen JT (1992) The methodology of multilocus DNA fingerprinting using radioactive or nonradioactive oligonucleotide probes specific for simple repeat motifs. In: Chrambach A, Dunn MJ, Radola BJ (eds) Advances in electrophoresis, vol 5. VCH, Weinheim, Germany, pp Fiala KL (1980) On estimating the primary sex ratio from incomplete data. Am Nat 115: Gerken T (2001) Kopulationen außerhalb des Paarbundes bei der Kohlmeise (Parus major) proximate Einflüsse und ultimate Faktoren. Ph.D. thesis, University of Bonn, Germany Glutz von Blotzheim UN, Bauer KM (1993) Handbuch der Vögel Mitteleuropas, Band 13/I, Passeriformes (4. Teil). Aula, Wiesbaden, Germany Griffith SC, Owens IPF, Thuman KA (2002) Extra-pair paternity in birds: a review of interspecific variation and adaptive function. Mol Ecol 11: Griffith SC, Örnborg J, Russell AF, Andersson S, Sheldon BC (2003) Correlations between ultraviolet coloration, overwinter survival and offspring sex ratio in the blue tit. J Evol Biol 16: Grindstaff JL, Buerkle CA, Casto JM, Nolan V, Ketterson ED (2001) Offspring sex ratio is unrelated to male attractiveness in dark-eyed juncos (Junco hyemalis). Behav Ecol Sociobiol 50: Hardy ICW (1997) Possible factors influencing vertebrate sex ratios: an introductory overview. Appl Anim Behav Sci 51: Hartley IR, Griffith SC, Wilson K, Shepherd M, Burke T (1999) Nestling sex ratios in the polygynously breeding corn bunting Miliaria calandra. J Avian Biol 30:7 14 Hunter, FM, Burke T, Watts SE (1992) Frequent copulations as a method of paternity assurance in the northern fulmar. Anim Behav 44: Jenni L, Winkler R (1994) Moult and ageing of European passerines. Academic Press, London, UK Jennions MD, Petrie M (2000) Why do females mate multiply? A review of the genetic benefits. Biol Rev 75:21 64 Kempenaers B, Verheyen GR, Dhondt AA (1997) Extra-pair paternity in the blue tit (Parus caeruleus): female choice, male characteristics, and offspring quality. Behav Ecol 8: King JR, Griffiths R (1994) Sexual dimorphism of plumage and morphology in the coal tit Parus ater. Bird Study 41:7 14 Koenig WD, Dickinson JL (1996) Nestling sex-ratio variation in Western bluebirds. Auk 113: Kokko H, Brooks R, McNamara JM, Houston AI (2002) The sexual selection continuum. Proc R Soc Lond B 269: Komdeur J, Pen I (2002) Adaptive sex allocation in birds: the complexities of linking theory and practice. Phil Trans R Soc Lond B 357: Krackow S (2002) Why parental sex ratio manipulation is rare in higher vertebrates. Ethology 108: Krackow S, Tkadlec E (2001) Analysis of brood sex ratios: implications of offspring clustering. Behav Ecol Sociobiol 50: Krackow S, Meelis E, Hardy ICW (2002) Analysis of sex ratio variances and sex allocation sequences. In: Hardy ICW (ed) Sex ratios: concepts and research methods. Cambridge University Press, Cambridge, UK, pp Krebs EA, Green DJ, Double MC, Griffiths R (2002) Laying date and laying sequence influence the sex ratio of crimson rosella broods. Behav Ecol Sociobiol 51: Leech DI, Hartley IR, Stewart IRK, Griffith SC, Burke T (2001) No effect of parental quality or extrapair paternity on brood sex ratio in the blue tit (Parus caeruleus). Behav Ecol 12: Lessells CM, Boag PT (1987) Unrepeatable repeatabilities: a common mistake. Auk 104:

9 571 Lessells CM, Mateman AC, Visser J (1996) Great tit hatchling sex ratios. J Avian Biol 27: Lubjuhn T, Sauer KP (1999) DNA fingerprinting and profiling in behavioural ecology. In: Epplen JT, Lubjuhn T (eds) DNA profiling and DNA fingerprinting. Birkhäuser, Basel, Switzerland, pp Lubjuhn T, Gerken T, Brün J, Epplen JT (1999) High frequency of extra-pair paternity in the coal tit. J Avian Biol 30: Magrath MJL, Green DJ, Komdeur J (2002) Sex allocation in the sexually monomorphic fairy martin. J Avian Biol 33: McCullagh P, Nelder JA (1989) Generalized linear models, vol 37. Chapman and Hall, London, UK Møller AP, Ninni P (1998) Sperm competition and sexual selection: a meta-analysis of paternity studies of birds. Behav Ecol Sociobiol 43: Molenberghs G (2002) Model families. In: Aerts M, Geys H, Molenberghs G, Ryan LM (eds) Topics in modelling of clustered data. Chapman and Hall/CRC, Boca Raton, Florida, pp Neuhäuser M (2004) Test for a biased sex ratio when the data are clustered. Env Ecol Stat 11: Oddie KR, Reim C (2002) Egg sex ratio and paternal traits: using within individual comparisons. Behav Ecol 13: Pen I, Weissing FJ (2000) Sexual selection and the sex ratio: an ESS analysis. Selection 1:59 69 Questiau S, Escaravage N, Eybert MC, Taberlet P (2000) Nestling sex ratios in a population of bluethroats Luscinia svecica inferred from AFLPTM analysis. J Avian Biol 31:8 14 Radford AN, Blakey JK (2000) Is variation in brood sex ratios adaptive in the great tit (Parus major)? Behav Ecol 11: Ramsay SM, Mennill DJ, Otter KA, Ratcliffe LM, Boag PT (2003) Sex allocation in black-capped chickadees Poecile atricapilla. J Avian Biol 34: Risch M, Brinkhof MWG (2002) Sex ratios of sparrowhawk (Accipiter nisus) broods: the importance of age in males. Ornis Fenn 79:49 59 Rosenfield RN, Bielefeldt J, Vos SM (1996) Skewed sex ratios in Cooper s hawk offspring. Auk 113: Rutstein AN, Gorman HE, Arnold KE, Gilbert L, Orr KJ, Adam A, Nager R, Graves JA (2005) Sex allocation in response to paternal attractiveness in the zebra finch. Behav Ecol 16: Saino N, Ellegren H, Møller AP (1999) No evidence for adjustment of sex allocation in relation to paternal ornamentation and paternity in barn swallows. Mol Ecol 8: SAS Institute Inc. (1989) SAS/STAT user s guide: Version 6, vol 1. SAS Institute Inc., Cary, North Carolina Schmoll T, Dietrich V, Winkel W, Lubjuhn T (2004) Bloodsampling does not affect fledging success and fledgling local recruitment in coal tits (Parus ater). J Ornithol 145:79 80 Schmoll T, Dietrich V, Winkel W, Epplen JT, Schurr F, Lubjuhn T (2005) Paternal genetic effects on offspring fitness are contextdependent within the extra-pair mating system of a socially monogamous passerine. Evolution 59: Sheldon BC, Ellegren H (1996) Offspring sex and paternity in the collared flycatcher. Proc R Soc Lond B 263: Sheldon BC, Andersson S, Griffith SC, Örnborg J, Sendecka J (1999) Ultraviolet colour variation influences blue tit sex ratios. Nature 402: Stauss M, Segelbacher G, Tomiuk J, Bachmann L (2005) Sex ratio of Parus major and P. caeruleus broods depends on parental condition and habitat quality. Oikos 109: Trivers RL, Willard DE (1973) Natural selection of parental ability to vary the sex ratio of offspring. Science 179:90 91 Verboven N, Käkelä M, Orell M (2002) Absence of seasonal variation in great tit offspring sex ratios. J Avian Biol 33: Westerdahl H, Bensch S, Hansson B, Hasselquist D, von Schantz T (1997) Sex ratio variation among broods of great reed warblers Acrocephalus arundinaceus. Mol Ecol 6: Westneat DF (1990) Genetic parentage in the indigo bunting: a study using DNA fingerprinting. Behav Ecol Sociobiol 27:67 76 Westneat DF, Clark AB, Rambo K (1995) Within-brood patterns of paternity and paternal behavior in red-winged blackbirds. Behav Ecol Sociobiol 37: Whittingham LA, Dunn PO (2000) Offspring sex ratios in tree swallows: females in better condition produce more sons. Mol Ecol 9: Williams GC (1979) The question of adaptive sex ratio in outcrossed vertebrates. Proc R Soc Lond B 205: Winkel W (1970) Hinweise zur Art- und Altersbestimmung von Nestlingen höhlenbrütender Vogelarten anhand ihrer Körperentwicklung. Vogelwelt 91:52 59 Winkel W (1975) Vergleichend-brutbiologische Untersuchungen an fünf Meisenarten (Parus spp.) in einem niedersächsischen Aufforstungsgebiet mit Japanischer Lärche Larix leptolepis. Vogelwelt 96:41 63 and Winkel W, Winkel D (1980) Zum Paarzusammenhalt bei Kohl-, Blau- und Tannenmeise (Parus major, P. caeruleus und P. ater). Vogelwarte 30: Winkel W, Winkel D (1997) Zum Einfluß der Populationsdichte auf die Zweitbrutrate von Tannenmeisen. Jahresber Inst Vogelforsch 3:29 Yamaguchi N, Kawano KK, Eguchi K, Yahara T (2004) Facultative sex ratio adjustment in response to male tarsus length in the varied tit Parus varius. Ibis 146: