Sex allocation and parental quality in tree swallows

Transcription

1 Sex allocation and parental quality in tree swallows Behavioral Ecology doi: /beheco/arn081 Advance Access publication 4 August 2008 Kira E. Delmore, a Oddmund Kleven, b Terje Laskemoen, b Susan A. Crowe, a Jan T. Lifjeld, b and Raleigh J. Robertson a a Department of Biology, Queen s University, Kingston, Ontario K7L 3N6, Canada and b Natural History Museum, Department of Zoology, University of Oslo, PO Box 1172 Blindern, NO-0318 Oslo, Norway In species with extrapair mating, females may choose a social mate who will contribute to the successful raising of their brood and a sire who can enhance the genetic quality of offspring. Female choice of social mate and genetic sire may thus be independent events, directed toward different types of reproductive benefits. Furthermore, if reproductive benefits derived through mating preferences differ for sons and daughters, a coupling between sex ratio adjustment and mate choice would be favored by selection. In this paper, we examined whether females adjust the primary sex ratio of offspring to the quality of the social male and/or the extrapair sire in a socially monogamous species with frequent extrapair mating, the tree swallow Tachycineta bicolor. Recent evidence suggests that females obtain compatible genes benefits through extrapair mating in this species. If genetic quality is more important for male than female fitness and contributes to higher variance in reproductive success among males than females, sex allocation theory would predict a male-biased sex ratio among extrapair offspring. However, we found no indication of sex ratio bias with paternity in mixed paternity broods. Instead, females skewed the sex ratio toward males in broods without extrapair paternity, which probably reflects a higher phenotypic quality of these males. Furthermore, we confirmed earlier findings that females in good condition produce male-biased broods. Thus, our results indicate that female tree swallows adjust the primary sex ratio to the phenotypic quality of their social mate and themselves and not to the genetic sire of their offspring. Key words: extrapair paternity, maternal condition, sex ratio, Tachycineta bicolor. [Behav Ecol 19: (2008)] INTRODUCTION Sex ratio theory predicts that, when the relative fitness costs and/or benefits of producing sons and daughters differ, selection should favor parents with the ability to bias the sex ratio of their offspring toward the sex with the higher relative reproductive value (Trivers and Willard 1973). Several factors may influence the relative reproductive value of sons and daughters, including the quality of the parental care they receive as young and the quality of their genes. In altricial birds, there is a large body of evidence that the phenotypic quality of nestlings at the time of fledging has a great impact on their performance later in life (e.g., Perrins 1965; Nur 1984; Birkhead et al. 1999). Likewise, there is increasing evidence that female birds, at least in some passerine species, enhance the genetic quality of offspring through extrapair mating (Griffith et al. 2002; Foerster et al. 2003; Stapleton et al. 2007; Fossøy et al. 2008). In polygynous or socially monogamous mating systems with extrapair paternity (EPP), variance in reproductive success is often greater in males than in females (reviewed in Whittingham and Dunn 2005). Accordingly, a successful son has the potential to produce many more offspring and provide greater fitness returns for parents than a successful daughter (Trivers and Willard 1973). If the future success of offspring is affected by fledgling quality (phenotypic and/or genotypic), parents might therefore benefit from skewing their brood sex ratio toward males when producing highquality offspring and toward females when producing offspring of inferior quality. Address correspondence to K.E. Delmore, who is now at Department of Biological Anthropology, University of Calgary, 2500 University Drive NW, Calgary, Alberta T2N 1N4, Canada. kedelmor@ucalgary.ca. Received 22 August 2007; revised 9 April 2008; accepted 15 May Ó The Author Published by Oxford University Press on behalf of the International Society for Behavioral Ecology. All rights reserved. For permissions, please journals.permissions@oxfordjournals.org In species where females engage in extrapair copulations (EPCs), a female can enhance the fitness prospects of her offspring in several possible ways. First, she can select a social partner who would contribute parental care and other resources to enhance the phenotypic quality of her offspring. Second, she can ensure a high genetic quality of her offspring through the selection of an extrapair mating partner with a favorable genotype (good genes or compatible genes). Both of the latter strategies have the potential to increase offspring quality (phenotypic or genotypic), leading to a third possible way for females to enhance the fitness prospects of her offspring: She can adaptively match the sex of her offspring with their quality. After the first strategy (enhanced phenotypic quality), a female should bias the sex ratio of her brood toward sons. After the second strategy (enhanced genotypic quality), a female should bias the sex ratio of extrapair offspring (EPO) toward sons. If fine-scale physiological control is not possible, females should bias the sex ratio of their brood in proportion to the number of EPO. Sheldon and Ellegren (1996) predicted in a study on collared flycatchers (Ficedula albicollis) that females should bias the sex ratio of EPO toward sons as these would be expected to inherit a sexy trait (good genes) from their fathers. A similar reasoning could be extended to also include extrapair mating for compatible genes, as long as the fitness increment from enhanced genetic quality of EPO is higher in sons than in daughters. Studies of sex ratio adjustments have been carried out in a large number of species but with contrasting results. Some have examined the influence of the quality of a female s social mate on brood sex ratios. Results from these studies have been mixed, with some producing positive results (e.g., collared flycatchers, Ellegren et al. 1996; blue tits [Cyanistes caeruleus], Sheldon et al. 1999; varied tits [Poecile varius], Yamaguchi et al. 2004) and others negative results (e.g., barn swallows [Hirundo rustica], Saino et al. 1999; great tit [Parus major], Radford and Blakey 2000; dark-eyed junco [Junco hyemalis],

2 1244 Behavioral Ecology Grindstaff et al. 2001; black-capped chickadees [Poecile atricapilla], Ramsay et al. 2003; black swan [Cygnus atratus], Kraaijeveld et al. 2007; common yellowthroats [Geothlypis trichas], Abroe et al. 2007). Studies have also examined the influence of paternity on both the sex of individual EPO and the sex ratio of broods with EPO. The majority of results from these studies have been negative (e.g., great reed warbler [Acrocephalus arundinaceus], Westerdahl et al. 1997; blue tit, Leech et al. 2001; tree swallow [Tachycineta bicolor], Whittingham and Dunn 2001; fairy martin [Petrochelidon ariel], Magrath et al. 2002; black-capped chickadee, Ramsay et al. 2003; black swan, Kraaijeveld et al. 2007; common yellowthroats, Abroe et al. 2007; but see blue tit, Kempenaers et al. 1997; coal tit [Periparus ater], Dietrich-Bischoff et al. 2006; redcapped robin [Petroica goodenovii], Dowling and Mulder 2006). We studied sex ratio adjustments in a well-established nestbox population of tree swallows. Tree swallows are socially monogamous but exhibit one of the highest levels of EPP in birds (50 87% of all broods contain EPO and extrapair sires father 36 53% of all nestlings; reviewed in Barber et al. 1996, see also Stapleton et al. 2007). EPP seems to increase offspring viability in tree swallows (Kempenaers et al. 1999; Stapleton et al. 2007) and results in high variance in male reproductive success (Kempenaers et al. 2001). Variance in female reproductive success is presumably low as there is little opportunity to increase their reproductive output numerically (egg dumping is rare; Dunn et al. 1994). Results from a previous study on a different population of tree swallows suggest that nestlings in better condition have an increased potential for survival and recruitment into the breeding population (McCarty 2001). Together, these facts suggest that in tree swallows, investment in high-quality sons should yield higher fitness returns than investment in daughters. In a previous study of tree swallows, Whittingham and Dunn (2000) found that maternal condition had a strong positive influence on the condition of sons (phenotypic quality) and that male-biased sex ratios were associated with females in better body condition. This relationship was reexamined in 2005 with additional years of data. Once again, females in better condition were found to produce more sons (Whittingham et al. 2005). During this same study, the relationship between female condition and offspring sex ratio was tested experimentally by clipping some flight feathers to reduce female body condition. Females with clipped feathers exhibited poorer body condition and had a lower proportion of sons in their broods (Whittingham et al. 2005). The above studies were all conducted on the same population of tree swallows, in Wisconsin. We replicated the correlative portion of these studies on a larger scale and in a different population of tree swallows, in Ontario. We will evaluate not only the influence of female body condition on brood sex ratios but also age (a potential indicator of breeding success; Risch and Brinkhof 2002) and number of mite holes in flight feathers (an indicator of parasite load, with the potential to reduce host fitness; Dunn et al. 1994; Thompson et al. 1997; Whittingham and Dunn 2000; but see Blanco et al. 2001). Purely replicative studies of this type (i.e., studies conducted on different populations from the same species) are being advocated by a number of authors (e.g., Palmer 2000; Griffith et al. 2003) as they are important for determining how repeatable empirical findings are between populations (Griffith et al. 2003). Replicative studies are also less vulnerable to selective reporting and provide more strength than quasireplicative studies (i.e., replication of previous studies using different species or systems) (Palmer 2000). In sum, we will test 4 predictions for adaptive sex ratio adjustment in tree swallows: If females adjust the brood sex ratio to the genotypic quality of offspring, 1) females should bias the sex ratio of EPO toward sons. If females lack the ability to match offspring sex with paternity for each individual young, we might rather predict that 2) females should bias the sex ratio of broods in proportion to the number of EPO in the brood. Alternatively, if females adjust brood sex ratio to the phenotypic quality of the offspring, we would predict that 3) females should produce male-biased broods when mated to a highquality social mate, and 4) that females in better condition should produce more sons. MATERIALS AND METHODS Field methods We conducted this study on a population of box-nesting tree swallows at the Queen s University Biological Station, near Chaffey s Lock, Ontario, Canada (44 34#N, 76 19#W), during the breeding season of This study was part of an ongoing research project on this population, which has been the subject of intensive studies over the past 3 decades (cf., Robertson and Rendell 2001; Sæther et al. 2005; Stapleton et al. 2007). The study area consists of 8 grids, ranging in size from 9 to 35 nest-boxes, and an additional 25 solitary nest-boxes distributed along the main road connecting the separate grids. The grids are located on hayfields bordered by mixed deciduous forests, and the nest-boxes on the grids are arranged with an interbox distance of 40 m along a row and 28 m across the diagonal. Of the 162 boxes in the study area, 96 were occupied by tree swallows. We checked nest-boxes every other day prior to the appearance of the first egg, at which point we visited the boxes daily until clutch completion. Twelve days postclutch completion, toward the end of the 14-day incubation period, we checked the boxes daily until hatching. Three days posthatching, we collected a small droplet (5 25 ll) of blood through brachial venipuncture from all nestlings (which we stored in lysis buffer for later genetic analyses) and measured nestling body mass (to the nearest 0.1 g using an electronic balance). Each nestling was toenail clipped for later individual identification. At this point, we also collected any unhatched eggs still present in the nest from which we sampled brain or muscle tissue. We stored tissues in alcohol for later genetic analysis. Twelve days posthatching, we banded nestlings with a Canadian Wildlife Service aluminum band, measured tarsus length (to the nearest 0.1 mm using a digital caliper), and obtained a second measurement of nestling body mass. We calculated nestling growth increment as the change in body mass from 3 to 12 days posthatching. Tree swallow nestlings reach their asymptotic body mass approximately days after hatching (Robertson et al. 1992). We caught adults using mist nests or nest-box traps (Stutchbury and Robertson 1986) primarily after the nestlings hatched. At capture, we banded adults with a Canadian Wildlife Service aluminum band and a color band (blue for males and red for females). We collected a small amount of blood through brachial venipuncture (which we stored in lysis buffer for later genetic analyses) and obtained measurements of body mass, tarsus length, and feather mite infestation. We estimated feather mite infestation by counting the number of mite holes present on feathers of the tail and both wings (primaries and secondaries) (Dunn et al. 1994). Feather mites are common ectoparasites of birds. We followed accepted practice in assuming that feather mites decrease the fitness of their hosts (e.g., Dunn et al. 1994; Thompson et al. 1997; Whittingham and Dunn 2000) but recognize that this assumption is open to question (e.g., Blanco et al. 2001). We also classified adult females and males as second year or after second year on the basis of plumage coloration and wing length (Rendell WB, Robertson RJ, unpublished data), respectively. We confirmed estimates from plumage coloration and wing length when possible using banding records from

3 Delmore et al. Sex allocation in tree swallows 1245 previous years. In each case, estimates from plumage coloration and wing length were correct. Molecular sex determination We extracted DNA from blood or tissue samples using a commercial kit (E.Z.N.A, Omega Bio-Tek, Norcross, GA) and determined sex by amplifying the CHD1 gene using primers P2 and P8 (Griffiths et al. 1998) by polymerase chair reaction (PCR) and digesting PCR products with HaeIII (Whittingham and Dunn 2000). We carried out PCR amplification on a GeneAmp 9700 Thermocycler (Applied Biosystems, Foster City, CA) in a total volume of 10 ll with the following reaction conditions: 6.5 ll of distilled water, 1.0 ll of103buffer, 0.5 ll of each primer, 0.3 ll of dntp (ABgene, Epson, UK), and 0.2 ll of DNA polymerase (DyNAzyme, Finnzymes, Espoo, Finland) added to 1 ll genomic DNA. Thermal cycling conditions included an initial denaturation at 94 C for 5 min and then 30 cycles of 94 C for 30 s, 50 C for 30 s, and 72 C for 45 s followed by 72 C for 7 min. A 5-ll aliquot of the PCR product was digested with 5 U HaeIII, 1.00 ll 103 buffer, and 3.5 ll distilled water in 37 C for 2 h. We separated digested PCR products by electrophoresis at 100 V for min on 3% agarose gels stained with ethidium bromide. Bands were visualized with UV light. To validate the molecular sex determination, we also applied it to 30 adults of known sex (15 males and 15 females). We were unable to determine the sex of 5 embryos and 2 nestlings (no amplification). Paternity analysis We analyzed paternity using 3 highly polymorphic microsatellite markers through PCR. Each 10-ll reaction consisted of about 30 ng of genomic DNA, 0.5 ll of each primer (forward primers were fluorescently dyed), 0.1 mm dntp mix (ABgene), and 0.2 units of DNA polymerase (DyNAzyme) in the manufacturer s buffer (final concentrations of 10 mm Tris HCl, 1.5 mm MgCl2, 50 mm KCl, and 0.1% Triton X-100). We ran PCR on a GeneAmp 9700 Thermocycler (Applied Biosystems). The PCR profile used consisted of an initial denaturing step at 94 C for 5 min, followed by 35 cycles consisting of 94 C for 30 s, X C (50 C for locus Aar4 and 55 C for loci HrU6 and HrU10) for X s (30 s for locus Aar4 and 40 s for loci HrU6 and HrU10), and 72 C for 30 s. The PCR profile was terminated with 72 C for 7 min followed by 4 C for 5 min. We sized PCR products using a capillary automated ABI 3100 sequencer (Applied Biosystems) and analyzed the data with GeneMapper v3.0 analytical software (Applied Biosystems). We calculated polymorphism of the microsatellite markers using CERVUS v3.0 (Kalinowski et al. 2007) (see Table 1). The combined exclusion probability for the 3 markers was higher than Null alleles were detected at 1 locus (HrU10) and mutations at 2 loci (HrU6 and HrU10) that are known to display relatively high mutation rates (Brohede et al. 2004, Anmarkrud et al. 2008). Hence, to resolve cases of parentage uncertainty due to a single allelic mismatch between the genotype of a young and the putative parent, we used an additional triplet of microsatellite markers (Ltr6, Pdol5, and Tbi81) to assign or exclude parentage. Detailed information about the PCR protocol and polymorphism of these latter markers is presented elsewhere (Stapleton et al. 2007). There is no evidence for rapid mate switching in this population of tree swallows (Kempenaers et al. 2001). As a result, any EPO identified are likely the result of EPCs. Statistical analysis A total of 471 DNA samples from 91 broods were collected, including 416 nestlings and 55 embryos from eggs that did not Table 1 Polymorphism among 3 microsatellite loci used for parentage analysis in tree swallows (Tachycineta bicolor) Locus (references) k n H O H E P ei P ej N e Aar4 (Hansson et al. 2000) HrU6 (Primmer et al. 1995) HrU10 (Primmer et al. 1996) Data on the number of alleles (k), number of genotyped adult individuals (n), observed heterozygosity (H O ), expected heterozygosity (H E ), probability of exclusion assuming no parents known (P ei ), probability of exclusion assuming one parent known (P ej ), and estimated frequency of null alleles (N e ) are presented. hatch or young that died in the nest. The sex ratio of unhatched eggs and nestlings (9 males: 13 females) that died before initial sampling and cannot be attributed to predation (i.e., at least one nestling was recovered from the nest) was not male biased (v 2 ¼ 0.73, P ¼ 0.39). Thus, differential mortality of male and female embryos or nestlings is not likely influencing brood sex ratios. Nevertheless, interpretation of this result requires caution as the sample size is low, reducing the power of this comparison. Accordingly, we restricted all analyses to broods in which sex could be determined for all eggs laid (i.e., primary sex ratio), which reduced our sample size to a maximum of 64 broods. We had blood samples of both parents and thus were able to determine paternity of every nestling, for 50 of those broods. Accordingly, the sample size for analyses of both paternity and primary sex ratio was reduced to a maximum of 50 broods. Sample size was further reduced in some analyses where measurements were missing, in particular, in analyses using offspring as many broods were lost due to predation or poor weather conditions and could not be measured on day 12 (n ¼ 29 broods for these analyses). We performed statistical analyses using JMP IN 6.0 (SAS, Cary, NC) and SPSS 12 (SPSS, Chicago, IL). We analyzed deviations of sex ratio variation from binomial expectation using James procedure for overdispersion, which yields a standard normal deviate (z) test statistic (Krackow et al. 2002). We compared the sex ratio of EPO with withinpair offspring using a conditional logistic analysis, which accounts for the potential dependency of sexes within broods (Molenbergs 2002). We examined the relationship between the number of EPO and the number of male offspring in broods with mixed paternity using Kendall s Tau as these variables were not normally distributed. We used generalized linear models with binomial errors and logit links to test the effect of 1) brood paternity (the presence or absence of EPO), 2) phenotypic traits of the social father (body condition, wing length, number of mite holes, and age), 3) maternal variables (body condition, number of mite holes, and age), and 4) environmental variables (laying date, clutch size at laying, and brood size at hatching; all of which may reflect the immediate parental investment conditions) on brood sex ratios. We initially ran these models separately. We then ran a final model including all significant predictor variables from the first models to examine their independence. We used brood as the unit of analysis, with the number of male offspring in each brood as the dependent variable and brood size as the binomial denominator. Initially, we included all predictor variables in the model. We then removed the least significant effect, producing a reduced model that was treated the same way until only significant effects remained. Once we had obtained the final model, we reentered excluded terms as well as interaction effects one by one to confirm that they did not explain a significant part of the variation. We used likelihood

4 1246 Behavioral Ecology ratio tests after removal of the terms from the maximal model to determine the significance of parameters. To obtain a measure of body condition for the mother and social father, we included both body mass and tarsus length in models (tarsus length controls for structural size). Capture date was also included in maternal models as the body mass of adult females declines over the season, from incubation to nestling period (Robertson et al. 1992, Whittingham and Dunn 2000; this study r ¼ 20.49, P, , n ¼ 77). We transformed female body mass, female tarsus length, female number of mite holes, male body mass, and male tarsus length to normalize their distributions (log 10, log 10, square root, cube root, and cube root, respectively). We used 1-way analyses of covariance (ANCOVAs) to compare the condition of 1) females with and without EPO and 2) mother and male offspring condition. Nest identity was included as a random variable in the latter comparison to control for environmental and parental effects. Body mass was used as the dependent variable in these comparisons, with tarsus length as a covariate. We used a panel regression to compare the body condition of nestlings in broods with EPO and those in broods without EPO, which allows us to control for both tarsus length (structural size) and nest identity (environmental and parental effects) (Donner and Cunningham 1984). Lastly, we examined the relationship between offspring sex and nestling characteristics (body mass 12 days posthatching and growth increment between 3 and 12 days posthatching) using linear mixed effects models with restricted maximum likelihood method. We ran separate models for each nestling characteristic. We included nest identity as a random effect in these analyses to control for environmental and parental effects. We restricted these analyses to broods containing both male and female offspring. RESULTS Variance in the overall offspring sex ratio (49.0% male; 359 offspring in 64 broods) was not significantly different from binomial expectation (z ¼ 1.59, P ¼ 0.11). In total, 48% (175/ 365) in 82% (55/67) of broods were sired by extrapair males. For the subsample of broods with data on primary sex ratio, extrapair fertilizations accounted for 48% (135/284) of the young in 82% (41/50) of the broods. EPO were equally likely to be male or female (conditional logistic regression; parameter estimate 6 standard error [SE] ¼ , v 2 ¼ 0.002, degrees of freedom ¼ 1,49, P ¼ 0.97). There was no relationship between the number of EPO and the number of male offspring in a brood (Kendall s T ¼ , P ¼ 0.94, n ¼ 50 broods). None of the phenotypic characteristics of social males explained variation in brood sex ratio (Table 2), but males without paternity loss had significantly more male-biased broods than those with paternity loss (63% vs. 46% male offspring; Table 2; Figure 1). Female body condition was also a significant predictor of brood sex ratio; the proportion of sons within a brood increased significantly with female body condition (Table 2). None of the other maternal variables were significant predictors of brood sex ratio nor were any of the environmental variables (Table 2). Brood paternity and female body condition were the only significant predictors of brood sex ratio identified in these separate generalized linear models. These variables were included in a final generalized linear model to determine if they reflected independent effects. Brood paternity lost significance when included with female body condition (i.e., when tarsus length and capture date were included in the model; Table 3). Nevertheless, brood paternity still showed a tendency toward influencing brood sex ratio (P ¼ 0.07; Table 3). In Table 2 Summary of separate generalized linear models examining brood sex ratio variation in relation to brood paternity, phenotypic traits of the social father, maternal variables, and environmental variables Predictor variables Parameter estimate SE v 2 P Brood paternity (n ¼ 50 broods) Intercept Brood paternity Phenotypic traits of social father (n ¼ 50 broods) Intercept Age Number of mite holes Wing length (mm) Body mass (g) Tarsus length (mm) Maternal variables (initial model, n ¼ 56 broods) Intercept Age Number of mite holes Body mass (g) Tarsus length (mm) Capture date Maternal variables (final model, n ¼ 57 broods) Intercept Body mass (g) Tarsus length (mm) Capture date Environmental variables (n ¼ 64 broods) Intercept Laying date Clutch size laying Brood size hatching Initial models are shown for each model. Where predictor variables were significant, final models (i.e., models following removal of nonsignificant effects by stepwise backward deletion) are also shown. Female body mass, female tarsus length, female number of mite holes, male body mass, and male tarsus length were all transformed in order to meet the assumption of normal distributions (log 10, log 10, square root, cube root, and cube root, respectively). Sample size is increased in the final model for maternal variables because removal of the nonsignificant predictor variables age and number of mite holes added an additional female to the model that was missing information for these 2 variables.

5 Delmore et al. Sex allocation in tree swallows 1247 Table 4 Linear mixed effects models with restricted maximum likelihood methods (nest identity used as a random effect) testing the effects of offspring sex on body mass 12 days posthatching and growth increment between days 3 and 12 posthatching in broods containing offspring of both sexes (n ¼ 29 broods) Factor Parameter estimate SE Degrees of freedom T ratio P Figure 1 Relationship between brood sex ratio (proportion of males) and brood paternity (broods without EPO contained no EPO and broods with EPO contained one or more EPO) in broods where sex was determined for all offspring. Columns denote mean values (6SE) of raw data. addition, when tarsus length and capture date were removed, brood paternity was a significant predictor of brood sex ratio (Table 3). Furthermore, a comparison of the body condition of females with and without EPO suggested that mean body condition was not significantly different between these 2 groups of females (females with EPO: g/mm; females without EPO: g/mm; 1-way ANCOVA, F 1,44 ¼ 0.50, P ¼ 0.48). If males with full paternity are betters fathers and females in good body condition are better mothers, we would expect the body condition of sons to be improved. A comparison between the body condition (body mass, controlling for tarsus length) of male nestlings in broods with EPO and those in broods without EPO, however, suggests that there is no significant difference Table 3 Summary of generalized linear models examining brood sex ratio variation in relation to predictor variables shown to be significant in separate generalized linear models (n 5 47 broods) Predictor variables Parameter estimate SE x 2 P Intercept Female mass (g) Female tarsus (mm) Female capture date Brood paternity Intercept Female mass (g) Brood paternity Two models are shown: 1 including female tarsus length and capture date to control for structural size and temporal variability and 1 with these nonsignificant variables removed. Sample size is reduced from 50 (the broods for which we have full paternity) because we lack information from 3 of the females for one or more of the variables. Body mass Intercept , Sex , Growth increment Intercept , Sex , between these 2 groups (panel regression, unstandardized coefficient ¼ 21.1, SE ¼ 1.04, t ¼ 21.04, P ¼ 0.30). However, a comparison between the body condition of mothers and male offspring revealed a significant positive relationship (1-way ANCOVA, F 1,26 ¼ 7.82, P ¼ 0.01). Increased cost (i.e., increased requirement of parental resources) of producing male offspring may be responsible for the observed sex ratio adjustments; if sons are more costly to produce, only parents in good condition should be able to afford their production. If sons fledged at a larger body mass than daughters, for instance, they would be expected to require more parental resources. Separate linear mixed effects models revealed that sex was a significant predictor of both body mass 12 days posthatching and growth increment between 3 and 12 days posthatching; males were larger and grew faster than their female nest mates (Table 4). DISCUSSION Evidence from our study suggests that female tree swallows adjust brood sex ratio to their parental capacity for rearing offspring: Broods were more male biased when the social male managed to ensure full paternity in the brood (prediction 3) and when the female was in good condition (prediction 4). Female tree swallows do not appear to adjust the brood sex ratio to the genotypic quality of offspring: EPO were equally likely to be male or female (prediction 1), and there was no correlation between the number of offspring that were male and the number of offspring resulting from EPCs in mixed paternity broods (prediction 2). We assume that males without paternity loss were of superior phenotypic quality. This is based on the fact that almost all female tree swallows seem to engage in extrapair mating (Barber et al. 1996; Stapleton et al. 2007; this study), and thus, males that manage to secure full paternity in their brood must be efficient sperm competitors or females do not engage in EPCs when mated to superior mates. Copulation data for most males in the present study reveal that those with full paternity copulated at higher rates than those with paternity loss (Crowe SA, Kleven O, Delmore KE, Laskemoen T, Nocera J J, Lifjeld JT, Robertson RJ, unpublished data) which means that full paternity might be explained by male virility alone without a need to invoke arguments about female choice. Identification of reliable cues to male phenotypic quality in the monomorphic tree swallow has been notoriously difficult (e.g., Kempenaers et al. 1999). A recent study conducted by Bitton et al. (2007), however, suggests that male age and plumage brightness may be indicators of male quality. Male age was not related to sex ratio adjustment in our study. Plumage brightness was not measured in our study but should be

6 1248 Behavioral Ecology included in any future studies examining the influence of paternal variables on brood sex ratio in the species. Maternal condition may also influence the phenotypic quality of offspring. Females in good condition biased the sex ratio of their broods toward sons and produced heavier sons. It should be noted that the latter results are consistent with findings of Whittingham and Dunn (2000) and Whittingham et al. (2005) on a different population of tree swallows, suggesting that condition-dependent sex ratio adjustment is repeatable between populations of tree swallows. It should also be noted that our measure of condition is rather indirect, as body mass recorded during the early nestling period may not reflect the female s physiological breeding condition at the onset of breeding, when the sex of eggs is determined. However, Whittingham et al. (2005) conducted an experimental study by reducing the flight performance of females at the onset of breeding through clipping of flight feathers. The treatment reduced their body condition and they produced more daughters than control females, suggesting a direct link between female parental capacity and the primary sex ratio. There is some evidence in tree swallows to suggest that body condition at fledging may be more important for future reproductive success in males than in females. As discussed in the introduction, variance in male reproductive success is much greater than that of females in tree swallows (Kempenaers et al. 2001). Results from our study, as well as other tree swallow studies (Whittingham and Dunn 2000; Whittingham et al. 2003; Whittingham et al. 2007), also suggest that male offspring may be more costly to produce: The asymptotic nestling mass of tree swallow sons tended to be greater than that of daughters as did the growth increment of sons between 3 and 12 days posthatching. A higher growth rate and higher asymptotic body mass in males than in females was also found in the Wisconsin population (Whittingham et al. 2007). These results suggest that the energetic demands required to produce tree swallow sons may be greater than those required for daughters (e.g., Fiala and Congdon 1983; Teather and Weatherhead 1988; Krijgsveld et al. 1998). Accordingly, sons may be more vulnerable to adverse environmental conditions making it beneficial for females to fine-tune their reproductive effort according to their own condition, producing more sons when they are in good condition and more daughters when they are in poor condition. This relationship has been found in a number of other bird species (e.g., American kestrels [Falco sparverius], Wiebe and Bortolotti 1992; lesser black-backed gull [Larus fuscus], Nager et al. 1999). In conclusion, results from our study suggest that female tree swallows adjust the primary sex ratio of their brood to the phenotypic quality of their social mate and themselves and not to the genetic sire of the offspring. Our findings contribute to the growing body of evidence suggesting that female birds may adjust the primary sex ratio to fine-tune their reproductive investments (e.g., Seychelles warbler [Acrocephalus sechellensis], Komdeur et al. 1997; lesser black-backed gull [L. fuscus], Nager et al. 1999; Eurasian kestrel [Falco tinnunculus], Korpimäki et al. 2000; house wren [Troglodytes aedon],whittingham et al. 2002; tree swallow, Whittingham et al. 2007). FUNDING Natural Sciences and Engineering Research Council of Canada Discovery grant (RGPIN/ ) to R.J.R.; Research Council of Norway (170853/V40) to J.T.L. We would like to thank Frode Fossøy and Hannah Munro for help in the field; Fran Bonier for helpful advice and comments on our manuscript; Bob Montgomerie for helpful discussions and comments; Tak Fung for statistical advice; and the Queen s University Biological Station staff for logistical support. Sampling for this study was conducted under Queen s University Animal Care permit R1, Canadian Wildlife Service (CWS) banding permit 10302, and CWS scientific capture permit CA0156. REFERENCES Abroe B, Garvin JC, Pedersen MC, Whittingham LA, Dunn PO Brood sex ratios are related to male size but not to attractiveness in common yellowthroats (Geothlypis trichas). Auk. 124: Anmarkrud JA, Kleven O, Bachmann L, Lifjeld JT Microsatellite evolution: mutations, sequence variation, and homoplasy in the hypervariable avian microsatellite locus HrU10. BMC Evol Biol. 8:138. Barber CA, Robertson RJ, Boag PT The high frequency of extrapair paternity in tree swallows is not an artifact of nestboxes. Behav Ecol Sociobiol. 38: Birkhead TR, Fletcher F, Pellatt EJ Nestling diet, secondary sexual traits and fitness in the zebra finch. Proc R Soc Lond B Biol Sci. 266: Bitton P-P, O Brien EL, Dawson RD Plumage brightness and age predict extrapair fertilization success of male tree swallows, Tachycineta bicolor. Anim Behav. 74: Blanco G, Tella JL, Potti J, Baz A Feather mites on birds: costs of parasitism or conditional outcomes? J Avian Biol. 32: Brohede J, Møller AP, Ellegren H Individual variation in microsatellite mutation rate in barn swallows. Mutat Res. 545: Dietrich-Bischoff V, Schmoll T, Winkel W, Krackow S, Lubjuhn T Extra-pair paternity, offspring mortality and offspring sex ratio in the socially monogamous coal tit (Parus ater). Behav Ecol Sociobiol. 60: Donner A, Cunningham DA Regression analysis in physiological research: some comments on the problem of repeated measurements. Med Sci Sports Exerc. 16: Dowling DK, Mulder RA Combined influence of maternal and paternal quality on sex allocation in red-capped robins. J Evol Biol. 19: Dunn PO, Robertson RJ, Michaud-Freeman D, Boag PT Extrapair paternity in tree swallows: why do females mate with more than one male? Behav Ecol Sociobiol. 35: Ellegren H, Gustafsson L, Sheldon BC Sex ratio adjustment in relation to paternal attractiveness in a wild bird population. Proc Natl Acad Sci USA. 93: Fiala K, Congdon J Energetic consequences of sexual size dimorphism in nestling red-winged blackbirds. Ecology. 64: Foerster K, Delhey K, Johnsen A, Lifjeld JT, Kempenaers B Females increase offspring heterozygosity and fitness through extra-pair matings. Nature. 425: Fossøy F, Johnsen A, Lifjeld JT Multiple genetic benefits of female promiscuity in a socially monogamous passerine. Evolution. 62: Griffiths R, Double MC, Orr K, Dawson RJG A DNA test to most birds. Mol Ecol. 7: Griffith SC, Örnborg J, Russell F, Andersson S, Sheldon BC Correlations between ultraviolet coloration, overwinter survival and offspring sex ratio in the blue tit. J Evol Biol. 16: Griffith SC, Owens IPF, Thuman KA Extra pair paternity in birds: a review of interspecific variation and adaptive function. Mol Ecol. 11: Grindstaff JL, Buerkle AC, Casto JM, Nolan V, Ketterson ED Offspring sex ratio is unrelated to male attractiveness in dark-eyed juncos (Junco hyemalis). Behav Ecol Sociol. 50: Hansson B, Bensch S, Hasselquist D, Lillandt B-G, Wennerberg L, von Schantz T Increase of genetic variation over time in a recently founded population of great reed warblers (Acrocephalus arundinaceus) revealed by microsatellites and DNA fingerprinting. Mol Ecol. 9: Kalinowski ST, Taper ML, Marshall TC Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment. Mol Ecol. 16: Kempenaers B, Congdon B, Boag P, Robertson RJ Extrapair paternity and egg hatchability in tree swallows: evidence for the genetic compatibility hypothesis? Behav Ecol. 10: Kempenaers B, Everding S, Bishop C Extra-pair paternity and the reproductive role of male floaters in the tree swallow (Tachycineta bicolor). Behav Ecol Sociobiol. 49:

7 Delmore et al. Sex allocation in tree swallows 1249 Kempenaers B, Verheyen GR, Dhondt AA Extrapair paternity in the blue tit (Parus caeruleus): female choice, male characteristics, and offspring quality. Behav Ecol. 8: Komdeur J, Daan S, Tinbergen J, Mateman C Extreme adaptive modification in sex ratio of the Seychelles warbler s eggs. Nature. 385: Korpimäki E, May CA, Parkin DT, Wetton JH, Wiehn J Environmental- and parental condition-related variation in sex ratio of kestrel broods. J Avian Biol. 31: Kraaijeveld K, Ming MA, Komdeur J, Mulder RA Offspring sex ratios in relation to mutual ornamentation and extra-pair paternity in the black swan Cygnus atratus. Ibis. 149: Krackow S, Meelis E, Hardy ICW Analysis of sex ratio variances and sex allocation sequences. In: Hardy ICW, editor. Sex ratios: concepts and research methods. Cambridge: Cambridge University Press. p Krijgsveld KL, Dijkstra C, Visser GH, Daan S Energy requirements for growth in relation to sexual size dimorphism in marsh harrier Circus aeruginosus nestlings. Physiol Zool. 71: Leech DI, Hartley IR, Stewart IRK, Griffith SC, Burke T No effect of parental quality or extrapair paternity on brood sex ratio in the blue tit (Parus caeruleus). Behav Ecol. 12: Magrath MJL, Green DJ, Komdeur J Sex allocation in the sexually monomorphic fairy martin. J Avian Biol. 33: McCarty JP Variation in growth of nestling tree swallows across multiple temporal and spatial scales. Auk. 118: Molenbergs G Model families. In: Aerts M, Greys H, Molenberghs G, Ryan LM, editors. Topics in modeling of clustered data. Boca Raton (FL): Chapman and Hall/CRC. p Nager RG, Monaghan P, Griffiths R, Houston DC, Dawson R Experimental demonstration that offspring sex ratio varies with maternal condition. Proc Natl Acad Sci USA. 96: Nur N The consequences of brood size for breeding blue tits II: Nestling weight, offspring survival and optimal brood size. J Anim Ecol. 53: Palmer AR Quasireplication and the contract of error: lessons from sex ratios, heritabilities and fluctuating asymmetry. Annu Rev Ecol Syst. 31: Perrins CM Population fluctuations and clutch-size in the great tit, Parus major L. J Anim Ecol. 34: Primmer CR, Møller AP, Ellegren H Resolving genetic relationships with microsatellite markers: a parentage testing system for the swallow Hirundo rustica. Mol Ecol. 4: Primmer CR, Møller AP, Ellegren H New microsatellites from the pied flycatcher Ficedula hypoleuca and the swallow Hirundo rustica genomes. Hereditas. 124: Radford AN, Blakey JK 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 Sex allocation in black-capped chickadees Poecile atricapilla. J Avian Biol. 34: Risch M, Brinkhof MWG Sex ratios of sparrowhawk (Accipiter nisus) broods: the importance of age in males. Ornis Fenn. 79: Robertson RJ, Rendell WB A long-term study of reproductive performance in tree swallows: the influence of age and senescence on output. J Anim Ecol. 70: Robertson RJ, Stutchbury BJ, Cohen RR Tree swallow; vol. 11. In: Poole A, Gill F, editors. The birds of North America, no. 1. Washington, DC: American Ornithologists Union. p Sæther B-E, Engen S, Møller AP, Visser ME, Matthysen E, Fiedler W, Lambrechts MM, Becker PH, Brommer JE, Dickinson J, et al Time to extinction of bird populations. Ecology. 86: Saino N, Ellegren H, Møller AP No evidence for adjustment of sex allocation in relation to paternal ornamentation and paternity in barn swallows. Mol Ecol. 8: Sheldon BC, Andersson S, Griffith SC, Örnborg J, Sendecka J Ultraviolet colour variation influences blue tit sex ratios. Nature. 402: Sheldon BC, Ellegren H Offspring sex and paternity in the collared flycatcher. Proc R Soc Lond B Biol Sci. 263: Stapleton MK, Kleven O, Lifjeld JT, Robertson RJ Female tree swallows (Tachycineta bicolor) increase offspring heterozygosity through extrapair mating. Behav Ecol Sociobiol. 61: Stutchbury BJ, Robertson RJ A simple trap for catching birds in nest boxes. J Field Ornithol. 57: Teather KL, Weatherhead PJ Sex-specific energy requirements of great-tailed grackle (Quiscalus mexicanus) nestlings. J Anim Ecol. 57: Thompson CW, Hillgarth N, Leu M, McClure HE High parasite load in house finches (Carpodacus mexicanus) is correlated with reduced expression of a sexually selected trait. Am Nat. 149: Trivers RL, Willard DE Natural selection of parental ability to vary the sex-ratio of offspring. Science. 179: Westerdahl H, Bensch S, Hansson B, Hasselquist D, von Schantz T Sex ratio variation among broods of great reed warblers Acrocephalus arundinaceus. Mol Ecol. 6: Whittingham LA, Dunn PO Offspring sex ratios in tree swallows: females in better condition produce more sons. Mol Ecol. 9: Whittingham LA, Dunn PO Survival of extrapair and withinpair offspring in tree swallows. Behav Ecol. 12: Whittingham LA, Dunn PO Effects of extra-pair and within-pair reproductive success on the opportunity for selection in birds. Behav Ecol. 16: Whittingham LA, Dunn PO, Clotfelter ED Parental allocation of food to nestling tree swallows: the influence of nestling behavior, sex and paternity. Anim Behav. 65: Whittingham LA, Dunn PO, Lifjeld JT Egg mass influences nestling quality in tree swallows, but there is no differential allocation in relation to laying order or sex. Condor. 109: Whittingham LA, Dunn PO, Nooker JK Maternal influences on brood sex ratios: an experimental study in tree swallows. Proc R Soc B Biol Sci. 272: Whittingham LA, Valkenaar SM, Poirer NE, Dunn PO Maternal condition and nestling sex ratio in house wrens. Auk. 119: Wiebe KL, Bortolotti GR Facultative sex ratio manipulation in American kestrels. Behav Ecol Sociobiol. 30: Yamaguchi N, Kawano KK, Eguchi K, Yahara T Facultative sex ratio adjustment in response to male tarsus length in the varied tit Parus varius. Ibis. 146: