Genetic similarity of mates, offspring health and extrapair fertilization in house sparrows

ANIMAL BEHAVIOUR, 2007, 73, 367e378 doi:10.1016/j.anbehav.2006.08.008

Genetic similarity of mates, offspring health and extrapair fertilization in house sparrows C H RIS TINE EDLY-W RIGHT*, P. L. S CH WA GM EYER* , PAT RIC IA G. PA RK ER† & DOUGLA S W. M OCK *

*Department of Zoology, University of Oklahoma, Norman yDepartment of Biology, University of Missouri at St Louis (Received 29 March 2006; initial acceptance 3 May 2006; final acceptance 24 August 2006; published online 8 Janurary 2007; MS. number: A10408)

High genetic similarity or dissimilarity between parents may result in viability consequences for offspring. However, because female choice of a social mate often reflects access to resources or male parental care, optimal genetic complementarity may be unattainable for social pairs. Thus, extrapair copulations may serve to increase offspring viability by allowing females to obtain sperm from males with more compatible genomes than those of social mates. We investigated whether parental genetic similarity influences offspring viability, including immunocompetence, in house sparrows, Passer domesticus. We also explored whether genetic similarity of social mates predicts the prevalence of young sired through extrapair fertilizations, and whether females enhance offspring viability by engaging in extrapair matings. We used DNA fingerprinting to estimate genetic similarity between social mates, and we assessed offspring viability using two measures of nestling immune response, the phytohaemagglutinin skin test and heterophil:lymphocyte ratios, plus nestling body mass and hatching success. We found no associations between the genetic similarity of parents and offspring immunocompetence, mass or hatching success; however, parental genetic similarity was positively correlated with clutch size. Genetic similarity of social mates also did not predict the frequency of extrapair young. Furthermore, maternal half-siblings within broods of mixed paternity did not differ in immunocompetence. Thus, our results provide no evidence for enhancement of offspring viability as an incentive for female engagement in extrapair copulations. Ó 2006 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Keywords: DNA fingerprinting; extrapair fertilization; house sparrow; mate choice; multiple mating; offspring viability and immunocompetence; Passer domesticus; sexual selection

In species that form social pairs, female choice of a social mate often reflects access to resources or parental care offered by males (Andersson 1994). However, in many such taxa, females are known to pursue extrapair copulations actively (e.g. visiting neighbouring territories and adopting solicitation postures: Kempenaers et al. 1992; Wagner 1993; Currie et al. 1998). The underlying causes of these extrapair matings are not well understood despite being a source of much recent study and debate. On the one hand, males that successfully sire offspring through

Correspondence and present address: C. Edly-Wright, 32 Andover Court, Bordentown, NJ 08505, U.S.A. (email: cedlywright@gmail. com). P. L. Schwagmeyer and D. W. Mock are at the Department of Zoology, University of Oklahoma, Norman, OK 73019, U.S.A. P. G. Parker is at the Department of Biology, University of Missouri at St Louis, 8001 Natural Bridge Road, St Louis, MO 63121, U.S.A. 0003e 3472/07/$30.00/0

extrapair copulation (EPC) clearly gain, unless their efforts to secure EPCs are balanced by negative consequences for reproduction with their social mates. On the other hand, because EPCs are presumed costly for females (reviewed in Westneat et al. 1990), and there is little evidence for most species that extrapair males offer material benefits (but see Gray 1997), it has been proposed that females engage in EPCs to gain genetic rewards (reviewed in Jennions & Petrie 2000). Females may accept extrapair matings with males possessing ‘good genes’ that enhance the viability or sexual attractiveness of offspring (Andersson 1994). Alternatively, genetic benefits may lie not in acquiring a mate with a generally superior genome per se, but rather a mate with a genome compatible to one’s own (Zeh & Zeh 1996, 2003; Mays & Hill 2004). Both categories of potential benefits to females predict positive effects on young sired through EPCs, and therefore, investigations of whether such effects exist provide insight into the

367 Ó 2006 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

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degree to which females are likely to profit from participating in extrapair mating systems. Genetic incompatibilities can result from any of several phenomena (Zeh & Zeh 1996, 1997; reviewed in Tregenza & Wedell 2000), including the degree of genetic similarity between mates. High genetic similarity between parents may reduce offspring viability via reduction in individual heterozygosity, which, in turn, can lead to expression of recessive deleterious alleles or eliminate heterosis effects. Genetic similarity of parental genomes may also result in lower genic diversity (e.g. at MHC loci; Brown 1997) or negative epistatic interactions between loci (Mitton 1993). Research documenting the consequences of inbreeding depression (reviewed in Charlesworth & Charlesworth 1987; Keller & Waller 2002) most plainly illustrates the costs of choosing a genetically similar mate. However, some degree of genetic similarity is, indeed, essential, and certain levels of inbreeding may be advantageous (Bateson 1983; Kokko & Ots 2006); thus, there may be a level of ‘optimal genetic similarity’ that exists between mates. The majority of avian studies that have used molecular techniques to explore the influence of mates’ genetic similarity per se on offspring viability (but see Hansson et al. 2001; Foerster et al. 2003; Schmoll et al. 2005a) have focused primarily on hatching success as a viability measure (Bensch et al. 1994; Kempenaers et al. 1996; Krokene & Lifjeld 2000; Cordero et al. 2004; Barber et al. 2005). Yet, viability benefits may extend into later life stages, including the nestling period and beyond. Furthermore, there is evidence that both the heterozygosity of avian offspring (Foerster et al. 2003) and their genic diversity (Hansson et al. 2001) can affect survival to breeding age, suggesting that parental genetic similarity may have important effects on later life stages. Of the studies that have investigated directly the effects of parental genetic similarity on offspring viability beyond the embryonic stage (e.g. in common shrews, Sorex araneus, Stockley et al. 1993; in coal tits, Periparus ater, Schmoll et al. 2005b), to our knowledge, none has examined offspring immunocompetence as a viability measure. However, the ability of an organism to mount an immune response to a pathogen may be an important determinant of survival. Indeed, a positive correlation between immunocompetence and further survival has been demonstrated for nestling (Christe et al. 1998) as well as adult birds (Saino et al. 1997b; Gonzalez et al. 1999; Soler et al. 1999), and genetic variability of immunocompetence in natural populations has been documented (reviewed by Read et al. 1995; Brinkhof et al. 1999). If optimal genetic similarity is unattainable with a social mate, EPCs may serve to increase offspring viability by allowing females to obtain sperm from males whose genes are more compatible with their own genes than are the genes of social mates (reviewed in Mays & Hill 2004; see also Masters et al. 2003). Yet, recent results addressing the relation between the genetic similarity of social mates and the prevalence of extrapair young (EPY) have been mixed (e.g. Ra¨tti et al. 1995; Krokene & Lifjeld 2000; Blomqvist et al. 2002; Freeman-Gallant et al. 2003; Eimes et al. 2004; Barber et al. 2005; Kleven et al. 2005).

If females do use EPCs to gain genetic benefits (either from good genes or compatible genomes), thereby enhancing offspring viability, extrapair young (EPY) should show greater viability than their within-pair young (WPY) half-siblings. Some evidence exists for viability advantages for EPY (Sheldon et al. 1997; Kempenaers et al. 1999; Johnsen et al. 2000; Foerster et al. 2003). In one case (Johnsen et al. 2000), the viability benefits have been attributed to genetic compatibility rather than to ‘good genes’ of extrapair sires, given that they were restricted to EPY and not experienced by the extrapair sires’ own offspring (the paternal half-sibs of EPY). However, other studies have failed to find differences in viability between within-pair young and EPY (Krokene et al. 1998; Strohbach et al. 1998; Whittingham & Dunn 2001; Kleven & Lifjeld 2004; Kleven et al. 2006) or have found the viability advantages to be restricted to particular environmental contexts (Schmoll et al. 2005a). This research aimed to delineate the relationships among genetic similarity of social mates, offspring viability and extrapair matings in the house sparrow, Passer domesticus. Specifically, we investigated whether genetic similarity of social mates influences offspring viability, including immunocompetence, and predicts the prevalence of EPY. In addition, we explored whether females enhance immunocompetence and general viability of their nestling offspring by engaging in extrapair matings. Finally, we also examined sources of variation in health measures between half-siblings in different nest environments (i.e. WPY produced from mate-changes throughout the breeding seasons). Good genes models emphasize the importance of paternal genes in shaping offspring viability, whereas genetic compatibility arguments propose that it is the interaction between maternal and paternal genotypes that has critical effects on offspring vigour (Zeh & Zeh 2003; Mays & Hill 2004). Nevertheless, measures of offspring health may be substantially influenced by maternal and paternal effects (see Cheverud & Moore 1994; Grindstaff et al. 2003). In song sparrows, Melospiza melodia, for example, Reid et al. (2003) found that cellmediated immunity of nestlings, but not of juveniles or adults, was significantly related to maternal inbreeding coefficient, and they suggested that this could arise from inadequate provisioning of eggs or young by inbred mothers. Moreover, strong extrinsic environmental influences on offspring health measures are widely acknowledged (e.g. Christe et al. 2000; Tella et al. 2000b). We used half-sib analyses among offspring of social mates to examine the magnitude of the variation in particular offspring traits that can be attributed to maternal factors (maternal effects and maternal genotype), paternal factors (paternal effects and paternal genotype), the interaction between the two, and the nest environment. Particular aspects of the life history and mating system of house sparrows make this species conducive to such investigations. House sparrows are sexually dimorphic, multibrooded and socially monogamous. Although some populations show a very low incidence of EPY (Griffith et al. 1999), EPY are more common in other populations (e.g. Wetton & Parkin 1991; Whitekiller et al. 2000; Stewart et al. 2006). Currently, there exists little documentation

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that female house sparrows encourage EPCs. Although forced EPC attempts (‘communal displays’) are conspicuous, fairly common, and resisted by females, female acceptance of unforced EPC solicitations is rarely observed (Wetton & Parkin 1991). If forced EPC attempts are conspicuous and frequently observed, but thought to be nearly always resisted successfully, and unresisted EPC attempts are rarely observed, then exploring potential incentives to females from EPC may shed some light on the role of females in the extrapair mating system. We applied molecular techniques to obtain measures of relative genetic similarity between social mates and to track extrapair paternity in the population. Viability of nestling house sparrows was assessed using two measures of immune response, the phytohaemagglutinin (PHA) skin test and heterophil:lymphocyte ratios (H:L ratios), as well as standard assessments of offspring viability (nestling mass, hatching success and fledging success). Positive correlations between the swelling response to a PHA immune challenge and nestling body condition (Saino et al. 1997a; Christe et al. 1998), survival prospects (Gonzalez et al. 1999; Soler et al. 1999) and longevity (Birkhead et al. 1999) have been demonstrated for other free-living birds. Because trade-offs may exist among the different facets of the immune system, measures from a single technique may not adequately reflect overall immunocompetence (Norris & Evans 2000). Therefore, in addition to the PHA skin test, we also compared H:L ratios between WPY and EPY as an alternative measure of immunocompetence, and related those offspring values to the genetic similarity of their parents. An increased H:L ratio is known to be a reliable indicator of mild to moderate physiological stress in birds (Gross & Siegel 1983; Maxwell 1993), including nutritional stress (Moreno et al. 2002). Thus, variation in H:L ratios between WPY and EPY may indicate unique immunological challenges experienced by WPY and EPY.

METHODS

Study Population We conducted this study during the breeding seasons of 2001 and 2002 at a site containing 38 nestboxes, located at North Base, University of Oklahoma, Norman. House sparrows had occupied boxes at the study site since 1994, and an earlier study of parentage at this and nearby sites revealed 20% of the young to be extrapair (Whitekiller et al. 2000). Nestboxes were censused twice weekly from March to August to determine the date that the first egg of each clutch was laid, eventual clutch size and number of chicks that successfully fledged. To determine the number of chicks that hatched from each clutch, we supplemented information from the regular biweekly censuses with additional checks as each clutch approached hatching. Blood samples were collected from putative parents (i.e. resident adult pair) for DNA extraction and screening. Eleven days following hatching, we weighed, collected blood from, and banded nestlings with U.S. Fish and Wildlife Service aluminium bands plus unique combinations of plastic colour bands for field identification.

Measures of offspring mass are based on a total of 528 offspring within 148 broods.

Genetic Analyses DNA methods We collected approximately 100 ml of blood from each individual by puncturing the brachial vein and using preheparinized capillary tubes to transfer the blood immediately into lysis buffer (50 ml of blood per 0.5 ml of Longmire’s solution) for DNA extraction and analysis (Longmire et al. 1988). Samples were inverted several times and refrigerated later on the day of collection. Multilocus minisatellite DNA fingerprinting, as well as paternity exclusion of all nestlings, followed methods in Mauck et al. (1995). Briefly, DNA was extracted using standard phenol/chloroform/isoamyl alcohol extraction. Samples of putative parents were loaded sufficiently often onto gels that they were not more than five lanes from each chick’s lane. Agarose gels were put through denaturing and neutralizing washes, then Southern blotted onto nylon (Nytran brand, Florham Park, New Jersey, U.S.A.), and hybridized with radioactively labelled Jeffreys’ probe 33.15 (Jeffreys et al. 1985). We visualized banding patterns on a Storm (Sunnyvale, California, U.S.A.) phosphorimager and the data were stored as digital images.

Parental exclusion Two scoring criteria were empirically determined for the study population. All bands in chicks’ lanes were compared against the banding patterns of the putative parents; any bands left unmatched in the chick’s lane were labelled ‘unattributable’ to these putative parents. In addition, the proportions of bands shared between putative parents and chicks were calculated as Dice’s index, D ¼ 2NAB/(NA þ NB), where NAB is the number of bands shared by both individuals (parent and offspring) and NA and NB are the number of bands in individual A (parent) and individual B (offspring), respectively (Wetton et al. 1987). Mean  SD band sharing between first-order relatives (parents and assigned offspring and full siblings) was 0.608  0.144 (range 0.39e0.93, N ¼ 127). Mean band sharing between nonrelatives (identified as chicks from different nests in separate parts of the study area) was 0.32  0.109 (range 0.02e0.47, N ¼ 145). Mean  SD number of bands scored per individual was 18.3  3.7 (range 10e29). Any chick with a band-sharing value of 0.53 or less with either parent (0.53 was the upper 95% confidence limit of the distribution of band-sharing values for nonrelatives) and two or more unattributable bands was considered to have been incorrectly matched with that putative parent. Whether the male parent, female parent, or both were excluded was determined by dyadic band-sharing values of chicks with each putative parent. Chicks for which at least one putative parent was excluded scored a mean  SD of 3.26  1.67 unattributable bands. The probability of mistakenly accepting a genetically unrelated individual as a parent was derived by calculating the probability that a nonrelative would share the

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residual bands left unattributed to the first parent, or 4.73  104. The probability of mistakenly accepting a close relative of an actual parent as a parent was calculated as 0.035 (Rabenold et al. 1991).

Band sharing between mates and random opposite-sex adults One of us (C.E.-W.) scored fingerprints for calculation of all band-sharing values between putative parents and between presumably random opposite-sex adults. Random dyads were generated by scoring banding patterns for unpaired male and female DNA samples run on the same gels. At the time of scoring, paternity of nestlings was unknown to the scorer. Bands were recorded as either present in the male only, present in the female only, or shared by both male and female. We excluded blurred bands at the bottom fourth of the gel. Ninety pairs of adults were rescored and intraobserver repeatability was high (ANOVA: F79,80 ¼ 11.34, P < 0.0001, R ¼ 0.89). Because clarity differences among gels might bias scoring, we tested whether the DNA gel number affected bandsharing values. Gel number did not predict variation in band sharing between social mates and random adults (ANOVA: F37,141 ¼ 1.27, P ¼ 0.1634). We estimated genetic similarity of 98 pairs of social mates by comparing their DNA fragment banding patterns and computing Dice’s index. A mean  SD of 18.7  4.23 bands per individual (N ¼ 196 dyad members) were scored. A mixed model with a random dyad effect showed that band sharing between social mates did not differ between years (restricted maximum likelihood estimates: F1,82 ¼ 0.08, P ¼ 0.7798), nor did it correlate significantly with the absolute number of bands scored (Pearson correlation: r96 ¼ 0.14714, P ¼ 0.1482). Genetic similarity of random opposite-sex dyads was measured also by computing Dice’s index for 81 pairs of adults. Band sharing between opposite-sex dyads differed between years (ANOVA: F1,79 ¼ 4.87, P ¼ 0.0303), but did not correlate significantly with the absolute number of bands scored (r79 ¼ 0.1296, P ¼ 0.2489).

Immunocompetence Heterophil:lymphocyte ratios The H:L ratio is generally regarded as a product of both the acquired and innate immune systems (Norris & Evans 2000). Heterophils are nonspecific phagocytizing cells involved in inflammatory reactions (Dieterlen-Lie`vre 1988). Lymphocytes are involved in the recognition and destruction of various pathogens (Maxwell 1993), and decreased numbers of these cells may indicate immunosuppression and associated susceptibility to viral infections (Siegel 1995). Thus, a nestling with a higher H:L ratio is assumed to be undergoing greater physiological stress than a nestling with a lower H:L ratio (Gross & Siegel 1983; Maxwell 1993). The H:L ratio has been recommended for ecological investigations because it has been shown to have a very small measurement error, and heterophil proportional counts are highly repeatable (Ots et al. 1998). In our study, H:L ratios were calculated for 409 nestlings from 119

broods. Five days after hatching, we collected a drop of blood from the femoral vein of each nestling and smeared it immediately onto a microscope slide. Slides were fixed in methanol for 5 min (the same day as collection) and later stained by immersion for 30 min in commercial Giemsa stain diluted 1:5 (v/v) with distilled water. White blood cells were identified and counted using a light microscope with an oil-immersion lens (1000; Lucas & Jamroz 1961). Only fields of the smear comprising an evenly distributed monolayer of cells were examined. A minimum of 100 white blood cells on each slide were counted and classified as either lymphocytes, monocytes, heterophils, eosinophils, or basophils. The H:L ratio was then calculated for each sample. All cell counts were performed by the same person (C.E.-W.).

T-cell-mediated immune response potential The PHA skin test provides a reliable measure of the response potential of circulating T cells to an injected mitogen (Goto et al. 1978; McCorkle et al. 1980) and has been routinely used in studies of several avian species (e.g. Smits et al. 1999; Johnsen et al. 2000; Tella et al. 2000a), including house sparrows (Gonzalez et al. 1999; Westneat et al. 2004). The mitogenic effect of PHA induces a dense perivascular accumulation of T lymphocytes and infiltration by macrophages and basophils (Stadecker et al. 1977; Goto et al. 1978; McCorkle et al. 1980); thus a higher PHA response indicates a stronger ability to produce a T-cellmediated immune reaction (Goto et al. 1978; McCorkle et al. 1980). Merino et al. (1999) investigated the potential for physiological stress resulting from the PHA test in nestling house martins, Delichon urbica, and concluded that the assay does not constitute a serious stressor for free-living animals, although it has been shown to elevate metabolic rate of adult house sparrows (Martin et al. 2003). We compared the mean mass of 11-day-old offspring in the years of this study ðX  SD ¼ 24:0  2:24 g; N ¼ 151Þ with the mass of offspring in the year before and after the study ðX  SD ¼ 24:2  3:32 g; N ¼ 138Þ, and found no difference (ANOVA: F1,287 ¼ 0.12, P ¼ 0.7265). PHA responses of 444 offspring from 124 broods were measured across the two seasons. Both differential responses to PHA injections of male and female nestlings (Tschirren et al. 2003; Chin et al. 2005; Dubiec et al. 2006) and the lack thereof (Johnsen et al. 2000; Kleven & Lifjeld 2004) have been reported for other passerines. We did not determine the sex of offspring, and while adult house sparrows show no sex differences in PHA response during the nonbreeding season (Navarro et al. 2003), whether sex differences in nestling PHA responses exist in this species is unknown. Ten days post-hatch, we administered a subcutaneous injection of PHA (40 mg of PHA dissolved in 40 ml of phosphate-buffered saline) in the patagium (wing web) of one randomly selected wing, producing a local inflammation (Chandra & Newberne 1977). The other wing received an injection of only 40 ml of phosphate-buffered saline as a control. The site of each injection was marked with an indelible pen to assure that swelling was measured at the location of the injection. One of us (C.E.-W.) conducted all wing measurements. Wing thickness was measured

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with a digital micrometre (Mitutoyo Series 293), three times per wing, immediately before injections on day 10 and again, three times per wing, approximately 24 h ðX  SD ¼ 23 h 27 min  1 h 32 minÞ after injections when we returned to weigh, band and take blood from the nestlings. Variation in the time, postinjection, at which PHA responses were measured occurred on days when multiple broods were to be processed. Repeated diurnally measured PHA responses among captive adult house sparrows have been shown to be stable for 24e72 h postinjection (Navarro et al. 2003). In a small passerine species, compression of the micrometre may distort the swelling, leading to inaccurate measurements. Therefore, once the micrometre’s rotating contact caused the wing skin to begin twisting, we relaxed just enough pressure for the skin orientation to return to its normal position, then recorded the thickness measurement (Smits et al. 1999). Despite this attempt to avoid these compressive effects, preliminary analyses indicated significant reductions across the three successive measurements, and this flattening effect increased with the magnitude of the swelling. Given that measurements of the same wing on the same day were highly repeatable (ANCOVA: treated wing before injection: F443,888 ¼ 23.45, P < 0.0001, R ¼ 0.88; treated wing after injection: F443,888 ¼ 95.01, P < 0.0001, R ¼ 0.97; control wing before injection: F443,888 ¼ 28.49, P < 0.0001, R ¼ 0.90; control wing after injection: F443,888 ¼ 28.50, P < 0.0001, R ¼ 0.90), we used only the initial measurement on day 10 and day 11 for further calculations of immune response. We defined ‘swelling’ as the measurement taken after the injection minus the measurement taken prior to injection. The difference in ‘swelling’ of the wing web between the PHA-inoculated wing and the control (wing-swelling response) is generally used as an index of the cell-mediated immune response potential (Goto et al. 1978; McCorkle et al. 1980). As in other studies (Saino et al. 1997a; Brinkhof et al. 1999; Kleven & Lifjeld 2004), PHA responses of individuals were associated with body mass, and we used the residuals from the linear regression of individual wing-swelling response on body mass as our measure of PHA response (F1,442 ¼ 14.65, N ¼ 444, P < 0.0001). We found no correlation between the two measures of offspring immunocompetence: PHA response and H:L ratio (Pearson correlation: r401 ¼ 0.03702, P ¼ 0.4587).

Statistical Procedures All statistical analyses were generated using SAS software, Version 8 of the SAS System for Windows (SAS Institute, Cary, North Carolina, U.S.A.). The data were pooled for both 2000 and 2001 breeding seasons. H:L ratios were logarithmically transformed to improve normality (Emerson & Stoto 1983), and the transformed data were used in all subsequent tests. We calculated linear mixed models (SAS Proc Mixed) to test the effects of parental band sharing on clutch size and nestling health measures (PHA response, H:L ratio, mass). Only WPY were included in these analyses. Because chicks within the same broods, as well as chicks with the same

parents but from different broods, were not independent of one another, models included a random ‘brood nested in pair ID’ effect. To examine the possibility of a unimodal curvilinear relationship between parental band sharing and offspring health measures (i.e. that there is an optimal degree of parental band sharing), we initially included a quadratic term of parental band sharing. Akaike’s Information Criterion (AIC, smaller is better) was used to determine the better fit model (linear or curvilinear). We used ANOVA to test the effect of parental band sharing on brood size prior to fledging and for the comparison of band sharing between social mates versus random dyads; in the latter analysis, we included number of lanes that separated the individuals within dyads as a covariate to adjust for differential proximity on the gel of the fingerprints of social mates versus random opposite-sex dyads. Also, because band sharing between random dyads differed significantly between years, we initially included year as a covariate, but eliminated this term from the final model because it was not significant and resulted in an overall poorer fit of the model. To examine the effect of band sharing between social mates and hatching success, we used logistic regression, and model fitting was performed using SAS %GLIMMIX macro for Version 8 using the events/trials syntax (Krackow & Tkadlec 2001; Kuss 2002). The data set was limited to broods in which all hatched nestlings were WPY. The response variable consisted of the ratio of the number of eggs that hatched (number of events) to clutch size (number of trials), and a random dyad effect was included. We also used logistic regression to examine whether band sharing between social mates predicted the prevalence of EPY (SAS %GLIMMIX macro for Version 8). Nestlings for which the resident female was excluded as the mother were dropped from the analysis. The response variable was EPY/brood size (at day 11 post-hatch), and dyad was fitted as a random effect. As with the models examining nestling health measures, we initially included and evaluated the significance of a quadratic term of parental band sharing in tests of parental band-sharing effects on hatching success and proportion of EPY. To analyse differences in nestling health measures between EPY and WPY, we calculated mixed models with fixed and random effects (SAS Proc Mixed). The data set was limited to maternal half-siblings within broods of mixed paternity. A ‘brood nested in pair ID’ effect, as well as an interaction effect between EPF status and ‘brood nested in pair ID’, were both fitted as random. We used Akaike’s Information Criterion (AIC, smaller is better) to determine whether to retain interaction effects in models. We also used random effects models (SAS Proc Mixed) to examine variation in nestling health measures among broods of maternal/paternal half-siblings that resulted from changes in social mates across breeding attempts. Brood means in health measures were used for these analyses, and the data sets were limited to broods of females that changed mates at least once within the two seasons (for analysis of maternal half-siblings) and males that changed mates at least once within the two seasons (for analysis of paternal half-siblings). Additionally, broods containing any EPY were excluded. For analyses

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Table 1. Significance tests for explanatory variables in mixed models analysing band sharing of social mates in relation to phytohaemagglutinin (PHA) skin response, heterophil:lymphocyte (H:L) ratio and mass of within-pair young

PHA response H:L ratio Mass

Band sharing

Brood (pair ID)

Time in season

F1,112¼0.41 P¼0.5227 F1,113¼0.10 P¼0.7523 F1,122¼0.66 P¼0.4194

Z¼4.01 P<0.0001 Z¼4.17 P<0.0001 Z¼4.22 P<0.0001

F1,121¼15.76 P¼0.0001 F1,121¼8.75 P¼0.0037 NS

of maternal half-siblings, female and ‘male nested in female’ effects were fitted as random. For analyses of paternal half-siblings, male and ‘female nested in male’ effects were fitted as random. For all mixed models and logistic regressions, degrees of freedom were calculated via the Satterthwaite method (Littell et al. 1996). We originally included time in season as a covariate in most mixed models because factors such as ambient temperature and prey availability change radically across the season (see also Westneat et al. 2004). Additionally, for tests of parental band-sharing and mate change effects, we originally included ‘time in season* band sharing’ and ‘time in season*male/female ID’ interaction terms, respectively. We used F tests to determine the significance of fixed effects. Excluding the explanatory variables in question, fixed effects that were not statistically significant were dropped from the models and the more parsimonious models were calculated. Results are reported for these models only. Means are presented with their standard deviations.

RESULTS Parentage data were available for 517 offspring within 146 broods from 103 different adult pairs, and these indicated that 80% (415/517) were offspring of the resident pair (i.e. WPY). An additional 17% (89/517) had been sired through extrapair fertilization (EPY), 2% (12/517) were excluded as offspring of the resident female, and only one nestling (<1%) was excluded as offspring of both putative parents. Forty-five per cent of broods (66/146) contained nestlings excluded as offspring of one or both parents. Mean band sharing between social mates (0.303  0.0795, N ¼ 98) did not differ significantly from the mean band sharing between random dyads (0.332 

Mass d F1,312¼11.80 P¼0.0007 d

N 347 115 415 137 319 110

chicks broods chicks broods chicks broods

0.0859, N ¼ 81), once the number of lanes separating the fingerprints of scored individuals was taken into account (ANCOVA: F1,177 ¼ 0.27, P ¼ 0.6027). Parental band sharing had no significant effect on offspring PHA response, H:L ratio or mass (Table 1). Band sharing between social mates was positively correlated with clutch size (F1,61.9 ¼ 5.43, P ¼ 0.023), accounting for about 9% of the variance, but it was not associated with hatching success (F1,59.5 ¼ 0.04, P ¼ 0.848), or brood size prior to fledging (F1, 77 ¼ 0.69, P ¼ 0.409). Moreover, band sharing between social mates did not predict the incidence of EPY (F1,84.2 ¼ 1.25, P ¼ 0.267). The average PHA response of EPY was greater than that of their maternal half-sibs (WPY) in just under half (22 of 48) of the broods with mixed paternity. Collectively, the mean PHA response of WPY from these broods was 0.010  0.5374 (N ¼ 122), while the mean PHA response of EPY was 0.0122  0.4343 (N ¼ 64); on a within-brood basis, the mean difference (EPYWPY) in PHA responses of maternal half-sibs was 0.003  0.5015 mm (95% CI: 0.145, 0.1498). This difference was not statistically significant (Table 2). Similarly, there also were no significant differences in mean H:L ratios of EPY and WPY within mixed-paternity broods (collective means: WPY: 2.43  2.271, N ¼ 121; EPY: 2.10  1.668, N ¼ 62; mean difference: 0.13  1.785; 95% CI: 0.649, 0.3872; Table 2) or in mean mass of EPY and WPY (WPY: 24.1  2.68, N ¼ 138; EPY: 24.3  2.04, N ¼ 73; mean difference: 0.09  1.76 g; 95% CI: 0.394, 0.5642). Comparisons of maternal half-siblings within different nests (i.e. offspring from different fathers and in different brood environments) revealed a significant difference in the mean brood mass of offspring from different male social mates of the same female, but no differences in PHA responses or H:L ratios (Table 3). Comparison of paternal half-siblings within different nests (i.e. offspring from

Table 2. Significance tests for explanatory variables in mixed models analysing differences in nestling phytohaemagglutinin (PHA) skin response, heterophil:lymphocyte (H:L) ratio and mass between maternal half-siblings in mixed-paternity broods

PHA response H:L ratio Mass

EPF: extrapair fertilization.

EPF status

Brood (pair ID)

F1,149¼0.08 P¼0.7824 F1,149¼0.49 P¼0.4861 F1,167¼0.33 P¼0.5673

Z¼3.05 P¼0.0011 Z¼2.26 P¼0.012 Z¼3.70 P¼0.0001

Time in season

Mass

Number of broods

NS

d

48

F1,45.3¼16.86 P¼0.0002 NS

NS

48

d

54

EDLY-WRIGHT ET AL.: GENETIC SIMILARITY AND NESTLING HEALTH

Table 3. Significance tests for explanatory variables in random effects models analysing variation in phytohaemagglutinin (PHA) skin response, heterophil:lymphocyte (H:L) ratio and mass between maternal half-siblings in different nest environments

PHA response H:L ratio Mass

Male (female)

Female

Residual

Z¼1.48 P¼0.0692 No variation

Z¼1.27 P¼0.1014 No variation

Z¼1.75 P¼0.0405

Z¼1.03 P¼0.1513

Z¼2.54 P¼0.0056 Z¼4.58 P<0.0001 Z¼3.04 P¼0.0012

different mothers and in different brood environments) showed no significant differences in the mean brood PHA responses, H:L ratios or mass of offspring from different female social mates of the same male (Table 4). Furthermore, neither maternal identity (Table 3) nor paternal identity (Table 4) accounted for significant levels of variation in brood PHA responses, H:L ratios, or mass. Residual variation in both comparisons of half-siblings was significant for all three health measures.

DISCUSSION We found no association between the genetic similarity of parents and three potential measures of nestling viability: PHA responses, H:L ratios and mass. Although we know of no similar avian studies relating the genetic similarity of mates to offspring immune response, a significant negative correlation between parental band sharing and body size of young has been reported for the common shrew, Sorex araneus, (Stockley et al. 1993). Additionally, in contrast to several avian studies that have found the degree of genetic similarity of social mates to be negatively correlated with hatching success (Bensch et al. 1994; Kempenaers et al. 1996; Krokene & Lifjeld 2000; Cordero et al. 2004), we found no such relationship in our study. It is possible that genetic similarity of parents is associated with deleterious effects on offspring fitness in house sparrows, but that the consequences of mating with a genetically similar individual are simply unrelated to these particular measures of immunity or successful embryonic development. For example, such effects may be expressed after the nestling period, in which case they could be detected only through offspring survivorship analyses. Nevertheless, this does not really tell us much about why several similar studies have found support for a relationship between genetic similarity and hatching success,

Time in season F1,25¼4.98 P¼0.0348 NS F1,31.2¼9.23 P¼0.0048

N 12 43 13 43 16 60

females broods females broods females broods

while we did not. There are no conspicuous differences in sample sizes: for example, Kempenaers et al. (1996) used data from about 60 blue tit, Cyanistes caeruleus, broods, which is virtually identical to our sample size, yet their very notable F ratio (>7.0) dwarfs ours (approximating zero). Moreover, their estimates of genetic similarity also were based on multilocus fingerprinting, and they scored a similar number of bands per individual. Obviously genetic similarity of parents accounts for much greater variation in hatching success in blue tits than it does in house sparrows, but we cannot assess whether this is due to greater variation among house sparrow parents in hatching success (stemming perhaps from exposure to a broader range of environmental conditions and/or biparental sharing of incubation) or differences between house sparrows and blue tits in the sensitivity of embryonic development to the genetic similarity of parents. Keller et al. (2002) found that negative effects of high genetic similarity (i.e. inbreeding depression) of Darwin’s finches were more likely to be detected under poor environmental conditions. We found a lack of parental genetic similarity effects on nestling health over a fluctuation of environmental conditions within a season (as indicated by nonsignificant ‘time in season*band sharing’ interaction terms). In contrast to other studies (Bensch et al. 1994; Ra¨tti et al. 1995; Kempenaers et al. 1996), we found a positive association between genetic similarity of social mates and clutch size, although we found no association between parental genetic similarity and brood size prior to fledging. If negative consequences of mating with a genetically similar individual do exist in our population, increased clutch size may mitigate such disadvantages. As in other similar avian studies (Kempenaers et al. 1996; Krokene & Lifjeld 2000; Charmantier et al. 2004; Schmoll et al. 2005b), band sharing between social mates did not predict the frequency of EPY in our population.

Table 4. Significance tests for explanatory variables in random effects models analysing variation in phytohaemagglutinin (PHA) skin response, heterophil:lymphocyte (H:L) ratio and mass between paternal half-siblings in different nest environments Female (male)

Male

Residual

No variation

H:L ratio

Z¼0.61 P¼0.2710 No variation

Mass

No variation

Z¼2.42 P¼0.0077 Z¼4.32 P<0.0001 Z¼5.47 P<0.0001

PHA response

Z¼0.10 P¼0.4603 Z¼0.69 P¼0.2462

Time in season NS F1,54.3¼4.10 P¼0.0478 NS

N 22 61 22 60 27 83

males broods males broods males broods

373

374

ANIMAL BEHAVIOUR, 73, 2

Other studies have produced results that differ in both directions. For example, pied flycatcher, Ficedula hypoleuca, pairs with lower band sharing had EPY significantly more often (Ra¨tti et al. 1995), as did tree swallows, Tachycineta bicolor (Barber et al 2005). But the opposite pattern was found for three species of shorebirds (western sandpiper, Calidris mauri, common sandpiper, Actitis hypoleuca and Kentish plover, Charadrius alexandrinus), with genetically similar parents having significantly more EPY (both extrapair paternity and quasiparasitism: Blomqvist et al. 2002). Band sharing of social mates also was positively associated with the incidence of EPY arising from extrapair fertilization in Mexican jays, Aphelocoma ultramarina, a species in which both a risk of inbreeding and deleterious consequences of inbreeding have been documented (Eimes et al. 2004). Likewise, major histocompatibility complex (MHC) similarity between mates predicted prevalence of EPY in first broods of Savannah sparrows, Passerculus sandwichensis (Freeman-Gallant et al. 2003). Differences in EPF rates among species do not seem likely to account for the distinction between studies supporting versus failing to support this relationship, and as above, there also are no obvious discrepancies in sample sizes that explain the differences between the studies. It is possible that an optimal level of genetic dissimilarity between house sparrow mates may exist, but may be more subtle than our genetic methods were able to detect. Yet, Freeman-Gallant et al. (2003) found that MHC band sharing covaried with overall genetic similarity as estimated by multilocus DNA fingerprinting. Moreover, Stewart et al. (2006) used microsatellite loci to estimate genetic similarity in a Kentucky population of house sparrows, and they also failed to find any effect of similarity on frequency of EPY. Nor did they establish differences in genetic similarity between social and extrapair mates. Alternatively, it is possible that the variation stems from species differences in the degree to which females are able to detect genetic similarity, or able to exercise choice among potential EPC partners. Mays & Hill (2004) have also suggested that evidence of mate choice based on genetic compatibility might generally be more likely to be found in monomorphic species than in strongly dimorphic species, given that compatibility-based choice should be associated with relatively lower variance in male reproductive success. Accordingly, they argue that evidence of mate choice predicated on good genes should be more prevalent in sexually dimorphic species with greater variance in male reproductive success. On the other hand, Neff & Pitcher (2005) noted that, if strong sexual dimorphism reflects a history of intense directional selection on males, additive genetic variation in fitness-related traits might be reduced, in which case it might become more advantageous for females to choose mates on the basis of compatibility rather than good genes. Our finding with respect to parental genetic similarity and the prevalence of EPY thus may pertain to current perspectives regarding when females are likely to choose mates on the basis of compatibility versus good genes. However, both types of mate choice predict greater viability of EPY, and we found no evidence that extrapair mating was associated with our measures of offspring

viability. There were no significant differences between the PHA responses, H:L ratios or mass of maternal halfsiblings within broods of mixed paternity. This is in agreement with studies of reed buntings, Emberiza schoeniclus (Kleven & Lifjeld 2004) and barn swallows, Hirundo rustica (Kleven et al. 2006); in both cases EPY and WPY did not differ in their responses to PHA. However, our findings contrast with those of Johnsen et al. (2000), who found that bluethroat, Luscinia svecica, EPY had significantly higher PHA responses than their maternal half-siblings in the same nest. We concur with Kleven & Lifjeld’s (2004) conclusion that there simply exist species differences in the relative immunocompetence of EPY, as measured by PHA responses. Thus, in house sparrows, and perhaps in other avian species such as the reed bunting and barn swallow, any viability advantages generated by extrapair fertilization may not be related to offspring immunocompetence or may manifest during stages other than the nestling period. There is some indication that EPY in other avian species may be more viable than WPY during embryonic and adult stages. For example, Foerster et al. (2003) found that EPY in blue tits were more heterozygous than maternal half-siblings in the same nest, and recruited fledglings were more heterozygous than nonrecruits. In addition, more heterozygous individuals had higher reproductive success and a more elaborate male secondary sex trait (crown colour). Still, in a number of avian studies, no differences have been found between EPY and WPY in terms of embryonic viability (Whittingham & Dunn 2001), number, mass and tarsus length of fledglings (Krokene et al. 1998; Strohbach et al. 1998; Kempenaers et al. 1999; Johnsen et al. 2000; Schmoll et al. 2005a), or in recruitment, first-year reproductive performance and mortality (Lubjuhn et al. 1999; Whittingham & Dunn 2001; Schmoll et al. 2003). Indeed, a recent meta-analysis of differences between WPY and EPY in measures such as recruitment and reproductive success found that, across six passerine species, indirect benefits from EPCs are consistently very weak (Arnqvist & Kirkpatrick 2005). This pattern, as well as the diversity among avian species in other aspects of extrapair mating systems, has led some recent authors to emphasize sexual conflict as a major determinant of extrapair mating behaviour and its outcomes (Westneat & Stewart 2003; Zeh & Zeh 2003; Arnqvist & Kirkpatrick 2005). Maternal half-siblings within broods of mixed paternity offer a powerful opportunity for detecting the significance of paternal genotype on offspring traits given that the nest environment is held constant. It has been further assumed that WPY and EPY within broods experience uniform maternal effects (e.g. Johnsen et al. 2000; Kleven & Lifjeld 2004). Failure to find differences between WPY and EPY in a particular trait therefore could occur if either (1) the trait is heritable, but much of the genetically based variation in the trait is attributable to variation associated with maternal genotypes (see Reid et al. 2003), or (2) little phenotypic variation is attributable to genetic differences between individuals. Our analyses of half-siblings resulting from mate changes of individuals across successive broods (Table 3) generally support the latter interpretation. After controlling for both maternal and paternal effects, as

EDLY-WRIGHT ET AL.: GENETIC SIMILARITY AND NESTLING HEALTH

well as any significant within-season effects, variation attributable to differences between broods of both maternal and paternal half-siblings in nest environment (i.e. residual variance) remained significant for all nestling health measures. Other studies also have shown particular aspects of the immune response of nestling birds to be highly sensitive to factors associated with ‘nest environment’ (e.g. Christe et al. 2000; Tella et al. 2000b); we note that environmental variation among house sparrow broods may be especially likely, given that their breeding season is long (encompassing approximately 4 months in our area) and spans a diversity of changes in food abundance and ectoparasite intensity. We additionally found no significant variation in mean H:L ratios or the mean PHA responses among broods of maternal half-siblings, and no effect of female identity on average mass, H:L ratios or PHA responses of paternal half-siblings. Broods of maternal half-siblings in different nests did vary in body mass, however. Given that body masses of EPY and WPY within the same nest were similar, this is most likely due to variation among males in their nestling provisioning. In summary, we found no associations between genetic similarity of mates and the viability of their nestlings or the hatching success of their clutches. However, clutch size increased with parental genetic similarity (although brood size prior to fledging was not associated with genetic similarity). Additionally, genetic similarity of social mates did not predict the occurrence of EPY. Thus, we found no evidence that female house sparrows engage in EPCs as a means of countering genetic similarity with their social mates. Furthermore, if females of this species participate in EPCs for either ‘good genes’ or ‘genetic compatibility’ advantages, the absence of detectable differences in health measures of EPY and WPY indicates that such advantages are not expressed via production of especially robust offspring. There may exist other benefits to female house sparrows from accepting EPCs, however, Wetton & Parkin (1991) suggested the advantage may lie in enhanced fertilization success, a possibility that is probably best approached experimentally.

Acknowledgments DNA fingerprints were produced in the Des Lee Laboratory of Animal Molecular Ecology at the University of Missouri, St Louis. We thank those involved in the genetics laboratory work, especially Charlotte Roy, Kelly Halbert, Noah Whitman, Jenny Bollmer, Cintia Cornelius and Sara Shrock. Thank you to John Farmer for advice and instruction regarding blood cell techniques and analysis. Statistical advice was provided by John Kolassa and Jorge Mendoza. We are grateful for those who assisted in the field: Letitia Posey, Terri Bartlett, Ellen Bolen, Lace Svec, Taryn West, Leslee Stone, Jennifer Alig and Melissa Moore. Two anonymous referees and Bill Searcy provided valuable comments on the manuscript. This work complied with the Guidelines for the Use of Animals in Research and was approved by the University of Oklahoma’s Animal Care and Use Committee (A3240-01). Funding for this research was provided by the National Science Foundation (IBN-9982661), George

Miksch Sutton Scholarship in Ornithology, M. Blanche Adams and M. Frances Adams Memorial Scholarship, University of Oklahoma Graduate Student Senate and the University of Oklahoma, Department of Zoology.

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