Extrapair copulations reduce inbreeding for female red-backed fairy-wrens, Malurus melanocephalus

Animal Behaviour 83 (2012) 857e864

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Extrapair copulations reduce inbreeding for female red-backed fairy-wrens, Malurus melanocephalus Claire W. Varian-Ramos*, Michael S. Webster 1 School of Biological Sciences, Washington State University, Pullman, WA, U.S.A.

a r t i c l e i n f o Article history: Received 5 October 2011 Initial acceptance 24 November 2011 Final acceptance 8 December 2011 Available online 28 January 2012 MS. number: A11-00789R Keywords: extrapair mating heterozygosity inbreeding avoidance Malurus melanocephalus red-backed fairy-wren relatedness

In many socially monogamous species, females copulate with and produce offspring sired by males other than their social mates, yet it remains controversial whether or how females benefit from these ‘extrapair’ copulations. Recently, it has been suggested that females might benefit if they are able to copulate with extrapair males that are genetically dissimilar to themselves, thereby potentially increasing the heterozygosity and/or reducing the level of inbreeding of the resulting offspring. However, empirical tests of this hypothesis have been criticized because a low number of molecular markers can lead to biased estimates of relatedness among individuals, and because all studies to date have been correlational and therefore unable to rule out potentially confounding factors. The red-backed fairy-wren is a bird with very limited dispersal, and hence the risks of inbreeding are high. We used a panel of microsatellite markers to examine paternity and relatedness between mates in this species, and also conducted an experiment that manipulated relatedness between a female and her social mate. Results from both approaches showed that females paired to genetically similar males were more likely to produce young sired by extrapair males, and that those offspring were less inbred (more heterozygous) than within-pair offspring. Thus, female fairy-wrens are able to avoid the potential costs of close inbreeding through extrapair copulations. Ó 2012 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

It is now clear that females of many socially monogamous species copulate with males other than their social mates (Griffith et al. 2002). It is generally thought that females benefit from production of young sired by these extrapair males, yet despite nearly two decades of work, the adaptive benefits of extrapair paternity to females remain unclear and controversial (Griffith et al. 2002; Westneat & Stewart 2003; Arnqvist & Kirkpatrick 2005; Schmoll 2011). Although females might gain direct benefits by mating with extrapair males (e.g. Gray 1997; Kempenaers et al. 1999; Li & Brown 2002), in most systems it is thought that females benefit indirectly through increased offspring quality (reviewed in: Jennions & Petrie 2000; Zeh & Zeh 2001; Griffith et al. 2002). This might occur if females choose extrapair mates that possess a particular gene or set of genes that impart some benefit to the offspring (Hamilton 1990; Westneat et al. 1990). However, studies of this ‘good genes’ hypothesis have produced mixed results (e.g. Schmoll et al. 2003; Gustafsson & Qvarnström 2006).

* Correspondence and present address: C. W. Varian-Ramos, Biology Department, College of William and Mary, P.O. Box 8795, Williamsburg, VA 23187, U.S.A. E-mail address: [email protected] (C. W. Varian-Ramos). 1 M. S. Webster is now at the Department of Neurobiology and Behavior, and Cornell Lab of Ornithology, Cornell University, W361 Seeley G. Mudd Hall, Ithaca, NY 14853, U.S.A.

More recently, the focus has turned to the idea that the interaction between the male and female’s genes can affect offspring fitness, and thus females might benefit from choosing extrapair mates with whom they are genetically compatible (Tregenza & Wedell 2000; Mays & Hill 2004; Kempenaers 2007). Although ‘genetic compatibility’ might involve a few specific genes (e.g. the major histocompatibility complex (MHC); see Hughes & Yeager 1998; Penn & Potts 1999; Milinski 2006; Thoß et al. 2011) or extranuclear elements (Zeh & Zeh 1996), more general genome-wide compatibility is likely to have important fitness consequences in many populations. In particular, overall genetic similarity (i.e. genetic relatedness) between parents has been shown to have strong effects on offspring fitness (e.g. Amos et al. 2001; Coltman & Slate 2003; Foerster et al. 2003; Mulard et al. 2009), to affect mate choice in species that do not form long-term pair bonds (e.g. Bull & Cooper 1999; Stow & Sunnucks 2004; Thuman & Griffith 2005), and also to affect social mate choice in species that do form pair bonds (Cockburn et al. 2003; Cohen & Dearborn 2004; Mulard et al. 2009). Genetic similarity might also affect extrapair mate choice, particularly in populations where social mate choice is constrained in some way, as in these cases extrapair mating can provide females with a ‘second chance’ to choose a genetically compatible mate (Brooker et al. 1990; FreemanGallant et al. 2006). Accordingly, genetic compatibility may be a strong force driving the evolution of extrapair mating behaviour in

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C. W. Varian-Ramos, M. S. Webster / Animal Behaviour 83 (2012) 857e864

some populations (Tregenza & Wedell 2002; Brouwer et al. 2010), particularly those with limited dispersal, such as cooperative breeders and island populations (Frankham 1998), because in these systems the chance of being socially paired with a closely related individual is relatively high (Richardson et al. 2004). Several studies have examined the effect of genetic relatedness on extrapair paternity in recent years, thanks to the increasing prevalence and ease of genetic tools to determine both relatedness and heterozygosity. However, many such studies use only a few loci to estimate relatedness and heterozygosity (e.g. Smith et al. 2005; Foerster et al. 2006; Edly-Wright et al. 2007), which limits the ability to detect biologically significant differences in relatedness or heterozygosity (Smith et al. 2005), and may also lead to biased results if the same markers are used to assess both parentage and heterozygosity (Wetzel & Westneat 2009). Moreover, all previous studies of the effects of relatedness on extrapair mating in wild populations have been correlative, and therefore, did not experimentally control for possible confounding factors such as male age and attractiveness, pair bond length, or other unknown factors (Griffith et al. 2002). An experimental test would allow the effects of these factors to be removed, but such experimental tests in wild populations are extremely challenging as it is difficult to manipulate the relatedness of pairs (but see Tregenza & Wedell 2002; Pryke et al. 2011). A related unresolved issue concerns the ways in which genetic similarity might affect extrapair mating. First, if overall heterozygosity is strongly related to individual fitness (reviewed in Hansson & Westerberg 2002), females should choose maximally dissimilar males in order to maximize offspring heterozygosity (Mays & Hill 2004). Under this ‘genetic dissimilarity hypothesis’, females that are socially paired to closely related males should be most likely to produce extrapair young (relative to other females), and the males that they choose as extrapair mates should be less related to them than expected by chance (Griffith et al. 2002; Tarvin et al. 2005). Alternatively, heterozygosity may not have a strong effect on offspring fitness except in the extreme, that is, when closely related individuals breed to produce highly inbred offspring with low fitness (Keller & Waller 2002; Kruuk et al. 2002; Hansson 2004; Spottiswoode & Møller 2004; Rodríguez-Muñoz & Tregenza 2009). If so, then females paired with closely related social mates might copulate with extrapair males to avoid the costs of inbreeding (Tregenza & Wedell 2000) rather than to maximize offspring heterozygosity. Under the ‘inbreeding avoidance hypothesis’, females that are paired to closely related males should produce more extrapair young than females that are paired to distantly related males, but the extrapair mates chosen may not be more distantly related to the female than an average male in the population (Tarvin et al. 2005). In this study we used both correlational and experimental approaches to study the genetic dissimilarity and inbreeding avoidance hypotheses in the red-backed fairy-wren, Malurus melanocephalus, a small Australian passerine. Red-backed fairy-wrens are socially monogamous and typically pair for life, but have very high rates of extrapair paternity, with over 50% of offspring being the result of extrapair copulations (Webster et al. 2008). This species is also a cooperative breeder, with some young males (‘auxiliaries’) remaining on their natal territory to assist their parents in raising subsequent broods. Moreover, like many other cooperative breeders (Hatchwell & Komdeur 2000), natal dispersal is thought to be extremely limited in this species (below). We used a panel of microsatellite markers to examine whether this limited dispersal leads to a high risk of inbreeding, and whether females use extrapair copulations to reduce this risk. In addition to these correlative analyses, we also conducted an experimental test of the inbreeding avoidance hypothesis by

removing breeding males from groups with and without auxiliaries to create new breeding pairs, each consisting of a female paired with her own son or with an ‘unrelated’ male from a neighbouring group. This experimental approach allowed us to examine the effects of partner relatedness on extrapair paternity rates while controlling for the effects of social male attractiveness, length of pair bond, male age, and other unknown factors, and we predicted that experimental females socially paired to their own sons would produce more extrapair offspring than would control females paired to unrelated males. METHODS Field Methods We conducted our research on a population of red-backed fairy-wrens at a long-term study site in the forest surrounding the reservoirs of the Herberton Shire on the Atherton Tablelands in Queensland, Australia (145
250 E, 17
220 S). Research was conducted during the breeding seasons (OctobereFebruary) of 2004e2007 (breeding seasons are designated by the year in which they ended). In each year we captured adults with mist nets to collect a small (ca. 30 ml) blood sample taken from the brachial vein for genetic analysis, and also to individually mark each bird with a numbered aluminium band (Australian Bird and Bat Banding Scheme) and a unique combination of three coloured leg bands. Group compositions and territory boundaries were determined through repeated observations of the birds in the field. We monitored all breeding attempts by groups on the field site. On the sixth day after hatching, nestlings were banded and a small (30 ml) blood sample was taken from the tarsal vein of each nestling for genetic analysis. Experimental Methods Removal experiments were conducted in 2006 (N ¼ 7) and 2007 (N ¼ 25) at a second study site approximately 15 km southwest of our long-term study site. For our experimental treatment, we captured and removed breeding males from groups with a male auxiliary. Auxiliary helpers usually have delayed plumage maturation and are brown rather than the bright red and black of most males. However, they are reproductively capable and occasionally sire young (Webster et al. 2008). The removed breeding males were driven approximately 10 km away and released in suitable habitat where fairy-wrens were present. No males returned within the same breeding season, although one male removed in 2006 did return to the study site in the subsequent year. Within hours of each breeding male’s removal, the group’s auxiliary assumed the breeding position, socially pairing with his own mother. Our observations confirmed that these former auxiliaries interacted with the breeding females and showed behaviours typical of dominant breeding males (see below; see also Karubian et al. 2011). For experimental control groups, we removed breeding males from groups without auxiliaries. Again, within a few hours a new male, usually an auxiliary male from a neighbouring group, moved in and assumed the breeding position. Occasionally (N ¼ 4) females re-paired with an older bright male. These pairings were excluded from further analysis as plumage colour and age also play a role in male reproductive success (Webster et al. 2008). Thus, new breeding males in all manipulated groups used for analysis were 1-year-old former auxiliaries with dull brown plumage; the new breeding males differed between treatments in terms of whether they were closely related to the breeding female (experimental) or not (control). To control for female breeding status, all removals were conducted when the group female was in the latter stages of nest building (N ¼ 19) and/or had begun to lay or incubate eggs (N ¼ 12).

C. W. Varian-Ramos, M. S. Webster / Animal Behaviour 83 (2012) 857e864

In both experimental and control treatments we destroyed the existing nest and collected the eggs, if present, to force the females to renest with the new mate. Females usually renested within a few weeks of the removal (mean  SE ¼ 18.2  1.3 days; range 7e31 days). Because of high predation rates (see Results), in 2007, we collected the eggs from the first nest of each experimental group after approximately 6 days of incubation to ensure sampling of offspring from as many removals as possible. Embryos were removed and frozen for later genetic analysis. Any subsequent clutches were allowed to hatch normally and blood samples were taken from the nestlings when they were 3 days old. Genetic Methods Blood samples were stored in lysis buffer and kept at 4
C until they were returned to the laboratory and processed. We extracted DNA from blood samples using a standard phenol chloroform procedure (Westneat 1990). Embryos were kept frozen until processing and extracted using DEasy blood and tissue kit from Qiagen. We genotyped all individuals using a panel of highly polymorphic microsatellite loci developed for other passerine species (Table 1) following methods outlined in Webster et al. (2008). Briefly, we amplified each locus using fluorescently labelled primers and standard PCR procedures and visualized the alleles using an automated sequencer. We used 10 microsatellites for the paternity analysis, which gave us a probability of 0.9998 of excluding a random male in the population as the sire of each offspring. We used the program CERVUS 2.0 (Marshall et al. 1998) to assign fathers to all sampled offspring based on genotypes. We evaluated all paternity calls from CERVUS using other available data including possible presence of null alleles, identity of sires, sires of nestmates. In most cases we accepted the sire with the highest likelihood score as assigned in CERVUS, but in some cases the other sources of data, such as social information, paternity of other nestlings in the same brood and knowledge of null alleles led us to assign paternity to a different male that was genetically compatible with the offspring but had a slightly lower likelihood score (see Webster et al. 2008 for details). To estimate relatedness between individuals we added two additional microsatellites to increase the accuracy of our Table 1 Microsatellites used in various analyses Locus

No. of alleles

HO

P(excl.)

Freq. null

Mcy1*,y,1 Mcy2*,y,1 Mcy3*,y,1 Mcy4*,y,1 Mcy7*,y,1 Msp4*,y,2 Msp6*,y,2 Ase9*,y,3 Cuu28*,y,4 Phtr3*,5 Smm1y,6 Smm7y,6

6 6 19 11 13 21 11 10 20 6 9 20

0.73 0.36 0.69 0.84 0.70 0.83 0.74 0.80 0.43 0.23 0.41 0.80

0.51 0.20 0.81 0.71 0.56 0.77 0.56 0.63 0.46 0.44 0.22 0.78

0.01 0.00 0.14 0.01 0.03 0.03 0.02 0.00 0.20 0.42 0.02 0.05

All microsatellites were used for measures of relatedness. * Used for paternity analysis. y Used for measure of heterozygosity. 1 Developed for the superb fairy-wren, Malurus cyaneus (Double et al. 1997). 2 Developed for the splendid fairy-wren, Malurus splendens (Webster et al. 2004). 3 Developed for the Seychelles warbler, Acrocephalus sechellensis (Richardson et al. 2000). 4 Developed for the Swainson’s thrush, Catharus ustulatus (Gibbs et al. 1999). 5 Developed for the willow warbler, Phylloscopus trochilus (Fridolfsson et al. 1997). 6 Developed from the southern emu-wren, Stipiturus malachurus (Maguire et al. 2006).

859

relatedness estimates. We used the program SPAGeDi (Hardy & Vekemans 2002) to calculate Queller & Goodnight’s (1989) R, which is a likelihood estimate of relatedness based on gene sharing (a score of 1 represents two maximally dissimilar individuals, a score of 1 indicates clones, and a score of 0 represents the average relatedness of two randomly chosen individuals in the population). For estimates of heterozygosity we excluded one microsatellite with a high frequency of null alleles, as such loci can lead to an artificially decreased estimate of heterozygosity. We used the remaining 11 microsatellites to calculate the standardized heterozygosity (SH), which is an estimate of heterozygosity weighted by the average heterozygosity at each locus (Coltman et al. 1999) and appears to be well correlated with other measures of heterozygosity and offspring performance (Amos et al. 2001). Spatial Methods To examine the spatial genetic structure in our population, we used the 120 individuals alive and present on our field site in October of 2004. Polygons were created for each territory from global positioning system (GPS) points collected during the field season. The spatial coordinates for each individual were determined from the geometric centre of the territory polygon, and as such, males, females and auxiliary males from the same group all had the same spatial coordinates. Spatial autocorrelation analyses were completed using GenAlEx (Peakall & Smouse 2006) following Double et al. (2005). These analyses calculate a spatial autocorrelation coefficient r for each of the specified distance classes. This coefficient reflects the average relatedness of individuals within the distance class (Smouse & Peakall 1999). Because the mean  SE distance between territory centres on our primary field site is 113.9  6.9 m (range 31.2e331.0 m), we used 55 m as our distance class to ensure that the first distance class included only withingroup comparisons. We examined relatedness out to 10 distance classes (550 m). Statistical significance was calculated using 1000 random permutations and 1000 bootstrapped estimates of r (Peakall et al. 2003). We completed these analyses for all adults and for males and females separately. Statistical Methods We compared the relatedness of females to their social mate for females that produced at least one extrapair offspring in a brood and for females that produced no extrapair offspring in a brood using a generalized linear mixed model (GLMM) assuming a normal distribution of relatedness scores and an identity link. We used presence of extrapair young in the brood as our fixed effect and female identity as a random effect to control for multiple sampling of some females. We also used only one randomly selected brood per femaleesocial male dyad to eliminate pseudoreplication, and all broods were complete (i.e. we determined paternity for every egg laid to ensure that we had accurate measurements of extrapair paternity). To compare the relatedness of the social mate and the extrapair mate to the female, we created a GLMM of the difference in relatedness score between social and extrapair mates (R(female / social male)  R(female / extrapair male)) with female as a random factor to control for multiple sampling of some females. Again we assumed a normal distribution of relatedness scores and used an identity link. The intercept of this model represents the average difference between the two relatedness values, and if it is significantly different from zero, then there is a significant difference in relatedness of social and extrapair mates. We used each femaleesocial mateeextrapair mate triad only once to control for pseudoreplication. Similarly, we used a GLMM with a normal distribution and an identity link to model the difference in

C. W. Varian-Ramos, M. S. Webster / Animal Behaviour 83 (2012) 857e864

heterozygosity between extrapair chicks and their within-pair half siblings (SHWPY  SHEPY), except in this case we used brood as our random effect to control for multiple comparisons within broods (e.g. if there is one within-pair chick and three extrapair chicks, then there are three comparisons in the brood). For our experiment we compared the proportion of extrapair young produced by experimental and control females with generalized linear model (GLM) with a binomial distribution and a logit link since our response variable was a proportion. Ethical Note All animals used in this study were handled and released as humanely as possible. Relocation of breeding males in the removal experiment represents an improvement over many removal experiments in which removed individuals are killed. Anecdotally, in a previous study involving red-backed fairy-wrens kept in outdoor aviaries, two males that escaped established breeding territories near the aviary site. This suggests that males are able to set up new territories and breed after relocation. Collection of clutches of eggs is similar in impact to predation that occurs commonly at our site. Females generally renest within a matter of weeks. All procedures were approved by Institutional Animal Care and Use Committee (protocol no. 03653) of Washington State University, the James Cook University Animal Ethics Review Committee (approval no. A1004) and the Queensland Government Environmental Protection Agency. Export of samples from Australia was approved by the Australian Government Department of Environment and Heritage. RESULTS

Genetic correlation (r)

860

0.3 0.2 0.1 0 −0.1 −0.2 55 0.3 0.2 0.1 0 −0.1 −0.2 55 0.3 0.2 0.1 0 −0.1 −0.2 55

(a) All adults

110

165

220

275

330

385

440

495

550

(b) Males only

110

165

220

275

330

385

440

495

550

(c) Females only

110

165

220

275

330

385

440

495

550

Distance (m) Figure 1. Correlogram plot of genetic correlation coefficients (r) for red-backed fairywrens at specified distances for (a) all adults, (b) males and (c) females that were known to be alive in the population in October 2004. In all plots, the solid line represents average r with error bars representing the bootstrapped 95% confidence interval. Dotted lines represent the permuted 95% confidence interval surrounding average relatedness of two random individuals (r ¼ 0). The female graph starts at 110 m because there was only one female present on each territory.

their within-pair half-siblings (t43 ¼ 2.10, N ¼ 85, P ¼ 0.041; Fig. 2c).

Spatial Patterns of Relatedness Natal dispersal was extremely limited, particularly in males, which led to a significant genetic structure within this population (Fig. 1). As a whole, the population showed significantly positive r values at 55 m, 110 m, 165 m, 275 m and 385 m. Analysing the sexes separately, males showed a similar pattern, with significantly positive r values out to 275 m, whereas females showed no significant spatial genetic structure. Paternity We completed paternity analysis on 322 chicks from 110 complete broods produced by 58 different females. Of these, 175 (54%) were the result of extrapair copulations, and 75 (68%) of all broods contained at least one extrapair chick. Extrapair paternity varied between years, ranging from 39% extrapair young in 2004 to 68% in 2006 (Table 2). Correlative Results Females that produced extrapair young were more closely related to their social mate than were females that produced only within-pair young (F1,5 ¼ 6.79, N ¼ 64, P ¼ 0.048; Fig. 2a). There was no significant difference in relatedness of females to their social mate and extrapair mates (t44 ¼ 1.74, N ¼ 65, P ¼ 0.089), although the effect was in the predicted direction (Fig. 2b). Relatedness of females to their extrapair mates did not differ from the relatedness of females to the average male in the population (t44 < 0.40, N ¼ 65, P ¼ 0.69), suggesting that females do not choose maximally dissimilar males as extrapair mates. We also found that extrapair young were more heterozygous than were

Experimental Results We conducted a total of 31 removals across the 2 years of the experiment, but some were discarded from the analysis: four females re-paired with an older (bright plumaged) neighbouring male (two experimental, two control), three females disappeared after the removal (two experimental, one control), six females either failed to produce a clutch prior to the end of our field season or the clutch was depredated prior to sampling (four experimental, two control), and in one case (control) we had no genetic sample from the replacement male. Of the 17 remaining successful removals, nine were experimental removals and eight were control removals. Our removals were successful at manipulating pair relatedness, as experimental pairs were more closely related (R ¼ 0.44  0.05) than were control pairs (R ¼ 0.05  0.05; t15 ¼ 6.52, P < 0.0001). After the removal, both related and unrelated replacement males were anecdotally observed displaying typical breeding behaviours, including duetting with the female, defending the territory and mate guarding (i.e. aggressively chasing away intruding males); nonbreeding auxiliaries do not typically display these behaviours. No

Table 2 Extrapair paternity rates of red-backed fairy-wrens over various years of the study Year

No. of young

No. of nests

No. of EPY (%)

No. of nests w/EPY (%)

2004 2005 2006 2007 Total

62 116 53 91 322

22 38 19 31 110

24 72 36 43 175

10 29 16 20 75

EPY: extrapair young.

(39%) (62%) (68%) (47%) (54%)

(45%) (76%) (84%) (65%) (68%)

C. W. Varian-Ramos, M. S. Webster / Animal Behaviour 83 (2012) 857e864

0.1

0.1

1.1 (b)

0.05 Relatedness

Relatedness

0.05

−0.05

−0.1

0

−0.05

No EPY

Some EPY

−0.1

(c)

Standardized heterozygosity

(a)

0

861

Social mate

Extrapair mate

1

0.9

0.8

0.7

WPY

EPY

Figure 2. (a) Relatedness (R from Queller & Goodnight 1989) of social males to female red-backed fairy-wrens that produced no extrapair young versus those that produced at least one extrapair young. Values are least squares means  SE from the GLMM controlling for multiple measures for females. (b) Relatedness (R from Queller & Goodnight 1989) of females to the social mate they cuckolded and the extrapair mate they choose. Values are means  SE controlling for multiple measures for some females. (c) Heterozygosity (standardized heterozygosity) of within-pair and extrapair half siblings. Values are means  SE controlling for multiple measures from broods. EPY: extrapair young; WPY: withinpair young.

copulations were observed during the course of the experiment. Actual copulations are rarely observed in this species (C.W.V.-R. observed two copulations in 5 years), and may occur in the predawn hours, as has been suggested in other species of fairy-wren (Double & Cockburn 2000). We found that experimental females paired to a closely related male produced a larger proportion of extrapair young than did control females paired to an unrelated male (F1,15 ¼ 5.37, P ¼ 0.035); of the 27 offspring produced by experimental pairs, only one was an incestuous within-pair offspring, whereas 8 of the 24 offspring produced by control pairs were withinpair (Fig. 3). Among the control pairs, those that produced within-

9 Control 8

Experimental DISCUSSION

Number of removals

7 6 5 4 3 2 1 0

pair young were less related (R ¼ 0.177  0.087) than those that did not produce within-pair young (R ¼ 0.082  0.045; F1,6 ¼ 6.99, P ¼ 0.038). Furthermore, none of the offspring were sired by the removed male through sperm storage, suggesting that the extrapair young represented new extrapair copulations occurring after the removal of the original male. We also examined our long-term data set to see whether there were any ‘natural experiments’ where a female was naturally widowed and ended up paired with her son. In the 8 years of our long-term monitoring of this population (1998e2007), there have been two cases where a son paired with his mother under natural conditions. In one case (1999) the pair produced two broods, both of which consisted entirely of extrapair young. In the second case (2007), no successful broods were produced. These data fit with the patterns seen in our experiment.

0

33

66

100

% EPY Figure 3. Distribution of extrapair paternity rates in groups of red-backed fairy-wrens from the experiment. Black bars: experimental groups; white bars: control groups. EPY: extrapair young.

In our correlational analysis we found that females that produced extrapair offspring were more closely related to their social mates than were females that produced only within-pair young, and that extrapair offspring were more heterozygous than were their within-pair half siblings. These results suggest that females reduce the potential costs of breeding with closely related males by engaging in extrapair copulations, and that these extrapair copulations result in potential benefits to the resulting offspring. This study thus joins a growing number of others in providing correlational support of the use of extrapair mating as an inbreeding avoidance strategy, including studies that show differences in cuckoldry rates based on relatedness (Blomqvist et al. 2002; Eimes et al. 2005; Tarvin et al. 2005; Freeman-Gallant et al. 2006; Jouventin et al. 2007; Blackmore & Heinsohn 2008; Cohas et al. 2008; Bergeron et al. 2011), differences in the relatedness of within-pair and extrapair mates (Tarvin et al. 2005; Lindstedt et al. 2007; Fossøy et al. 2008), and differences in offspring heterozygosity (Foerster et al. 2003; Tarvin et al. 2005; Rubenstein 2007; Stapleton et al. 2007; Fossøy et al. 2008).

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Correlational studies, however, can be confounded by a number of factors that might be associated with both genetic relatedness and likelihood of extrapair paternity, such as territory quality or male age. Experimental tests that control for such factors are therefore necessary to understanding the evolution of mating behaviours (Griffith et al. 2002), and this is the first study to experimentally test the inbreeding avoidance hypothesis in a wild population. Our experimental results strongly support the findings of the correlational analyses, as females experimentally paired to closely related males had higher rates of extrapair paternity than did females experimentally paired to unrelated males. In this experiment all replacement males were 1 year old, had dull brown plumage, were former auxiliaries, and had just formed new social pair bonds with the group female in that season, but experimental pairs were significantly more related than control pairs. Moreover, anecdotal observations indicated that the experimental pairs were true breeding pairs, as the mothereson pairs, as well as control pairs, showed all of the behaviours associated with social mates in this species, including duetting and aggressive mate-guarding behaviour by the new breeding male. Unfortunately, no quantification of pair male behaviour was made in this study. A remaining confounding factor is that all experimental pairs were by necessity from territories that had auxiliary males at the start of the breeding season. Only two of eight control pairs had auxiliary males on the territory at the start of the breeding season, so it is possible that the same factors that influence the presence of auxiliaries (e.g. territory density or territory quality) may also influence extrapair paternity. However, our finding that relatedness was associated with extrapair paternity rate even among control pairs suggests that pair relatedness plays an important role in genetic mate determination. Extrapair mating by females socially paired to genetically similar males might reduce two different potential costs of inbreeding. First, inbred individuals have reduced fitness in numerous wild populations of animals (reviewed in Keller & Waller 2002). Second, males may be partially behaviourally (e.g. reduced copulations) or physiologically (e.g. low sperm counts) inhibited from reproduction when paired to closely related females, which would lead to low reproductive output for the breeding female. In this study, one offspring was produced from a pairing between a mother and a son, so at least some copulations must occur between closely related pair mates. Nevertheless, if such incest avoidance mechanisms are operating, females might avoid the cost of reduced fertility through extrapair copulations (Sheldon 1994). It is important to note that no offspring in either our experimental or our control treatments were sired by the removed male, suggesting that storage of older sperm is probably not responsible for the results we obtained. Our results suggest that some mechanism of kin discrimination is operating, but generally it is unknown how birds assess their relatedness to other individuals. For our experiment, it is possible that social association between the breeding female and her philopatric son allowed the female to identify her son as a close relative (e.g. Komdeur et al. 2004). However, it is unlikely that this mechanism can explain our correlational results, as natural mothereson pairings are very rare under natural conditions. Thus, it seems that females must have some other unknown mechanism (e.g. olfactory cues: mammals: Krackow & Matuschak 1991; Wedekind & Füri 1997; reptiles: Olsson et al. 2003; fish: Mehlis et al. 2008) by which to assess relatedness to their social mate and adjust extrapair mating behaviour accordingly. Alternatively, females may not alter copulation patterns according to relatedness (e.g. all females may engage in extrapair copulations), but rather postcopulatory mechanisms may result in fewer embryos being sired by closely related males (Tregenza & Wedell 2002; Kawano et al. 2009). In fact, a reduction in the ability of closely related or incompatible sperm to

fertilize eggs successfully (e.g. Pryke et al. 2011) could explain both the correlative and experimental results observed in our study. In either case, extrapair copulations can serve as a mechanism whereby females avoid the costs of inbreeding with closely related social mates. Although female fairy-wrens appear to avoid the costs of close inbreeding via extrapair paternity, they do not appear to choose maximally dissimilar males as extrapair mates (e.g. Amos et al. 2001), as extrapair mates were not less related to the female than the average male in the population. This suggests that females avoid close inbreeding rather than maximize offspring heterozygosity. Alternatively, females may be constrained in their ability to assess relatedness to males that they do not interact with frequently, or may face constraints on their ability to sample extrapair males (Mulder et al. 1994), and thus would be constrained to choosing extrapair mates at random from the population (Tarvin et al. 2005) or based on some other factor, such as plumage colour (Webster et al. 2008). It is also possible that the genetic relatedness measured here with microsatellites in correlated with genetic differences in a particular gene or set of genes such as MHC. Birds have been shown to choose mates based on MHC diversity (Richardson et al. 2005; Griggio et al. 2011; Promerova et al. 2011) and dissimilarity (Freeman-Gallant et al. 2003). However, further study is needed to determine whether genetic compatibility at specific genes influences extrapair mating decisions in the redbacked fairy-wren. Populations with very low natal dispersal distances, such as seen in many cooperatively breeding animals and/or island populations, should be at relatively high risk of inbreeding relative to species with high natal dispersal (e.g. Kleven & Lifjeld 2005; Fossøy et al. 2008). The low rates of dispersal observed in red-backed fairywrens leads to greater risk of social pairing of relatives, particularly among older females. Although individuals can potentially avoid inbreeding through social mate choice, when social mate choice is severely constrained, then extrapair mating is an alternative method of inbreeding avoidance (Freeman-Gallant et al. 2006). Our removal experiments showed that social pairing occurs rapidly in our study species, and this suggests that females may have little opportunity to assess potential social mates, possibly resulting in a higher incidence of pairing between related individuals. Interestingly, incestuous social pairings are more common in other species of fairy-wrens than in the red-backed fairy-wren (Brooker et al. 1990; Cockburn et al. 2003), and these species have been shown to have some of the highest extrapair paternity rates of any animal (Brooker et al. 1990; Mulder et al. 1994). The extremely high rates of extrapair paternity observed in this genus may be, in part, caused by the need to avoid close inbreeding (Brooker et al. 1990). This study provides compelling correlative evidence for the use of extrapair mating as an inbreeding avoidance mechanism, as well as the first experimental test of this hypothesis in a free-living population, and provides further evidence that genetic compatibility is an important force driving the evolution of extrapair mating in many socially monogamous species. Acknowledgments We thank our numerous field assistants from Team Wren and our WUndergrad lab techs. Special thanks to Willow Lindsay, Melissah Rowe and Jordan Karubian for assistance with the removal experiment. We also thank the members of the Webster and Schwabl labs for critical advice and support. Thanks to Tim Daniel, Jane Hart, Tom and Coral Risley, Brad Congdon and James Cook University for field support and logistics. We are grateful to the Australian and Queensland governments for permission to conduct this research. Principal funding support was provided by the

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