Extra-pair paternity is not driven by inbreeding avoidance and does not affect provisioning rates in a cooperatively breeding bird, the noisy miner (Manorina melanocephala)

Extra-pair paternity is not driven by inbreeding avoidance and does not affect provisioning rates... Abstract In many socially monogamous bird species, both sexes regularly engage in mating outside their pair bond. Although the benefits of extra-pair (EP) mating behavior are clear and well established for males, such as an increase in the number of sired offspring, the benefits of EP mating behavior to females are less clear. A dominant theory for the incidence of EP mating is that socially monogamous females can improve the genetic quality of their offspring and avoid the costs of inbreeding through EP mating. In addition, in cooperatively breeding species, the theory of “parental care” predicts that females obtain additional help for their offspring through EP matings. Here, we examined evidence for both the inbreeding avoidance and parental care hypotheses in the cooperatively breeding noisy miner (Manorina melanocephala). Overall, EP mating occurred in 27% of broods, with 14% of offspring sired by males other than the identified pair-bonded male. There was a strong tendency to avoid pairing with genetically related individuals, with 86% of breeding pairs being significantly less related to each other than the general population. The occurrence of EP paternity was independent of the degree of relatedness between the pair. Provisioning patterns in relation to EP mating was not consistent with the “parental care” hypothesis and EP males did not contribute to the care of broods. These results demonstrate that in this system, there is no evidence that EP mating might function as a mechanism to reduce the costs of inbreeding depression or to gain benefits of extra helpers. INTRODUCTION Almost decades of developments in parentage analysis have confirmed a higher complexity and diversity of mating systems in birds than those predicted by social bonds alone, demonstrating that in most species females do not mate exclusively with their social partners, but rather also frequently copulate with males outside their social bond (Birkhead 1998). This phenomenon, referred to as extra-pair (EP) mating, is widespread among bird species. For example, only 10% of socially monogamous songbirds are found to be genetically monogamous (reviewed in Griffith et al. 2002). EP mating has broad implications and might influence mate choice as well as sexual conflicts and speciation (Bretman and Tregenza 2005). Although the mechanism and evolution of EP mating in birds have been the central focus of many recent studies, how EP mating evolves and what benefits might be accrued still remain a topic of considerable debate (reviewed in Griffith et al. 2002; Westneat and Stewart 2003; Akçay and Roughgarden 2007). Although EP mating enhances the reproductive opportunities for males, the benefits that females receive are less clear given that EP mating typically does not enhance fecundity (Møller and Birkhead 1994). Furthermore, mating with multiple males can impose significant costs to breeding females (Cornell and Tregenza 2007), such as aggression (Valera et al. 2003) or decreased parental care from her pair-bonded male (Arnqvist and Kirkpatrick 2005), as well as increased exposure to parasites and pathogens (Martinez-Padilla et al. 2012). Despite these costs, the near ubiquity of EP matings in the majority of taxa suggests that females should receive indirect and/or direct compensatory benefits to support this behavior, although the assumption that EP mating is adaptive might be incorrect under some circumstances (e.g. forced copulations controlled by males; Akçay et al. 2012). As a result, many hypotheses have been developed to explain what benefits females might gain for polyandrous mating behavior (Griffith et al. 2002). One of these hypotheses suggests that females use EP mating as a mechanism to avoid inbreeding and to gain an indirect benefit of improved offspring fitness (Griffith et al. 2002; Westneat and Stewart 2003; Akçay and Roughgarden 2007). For instance, inbreeding can cause negative effects on offspring traits such as survival or immunity (Keller and Waller 2002; Reid et al. 2011; Hemmings et al. 2012; Gohli et al. 2013) or even egg hatchability (Kingma et al. 2013). Therefore, EP mating with genetically dissimilar males might improve the genetic diversity of offspring (Kempenaers 2007) and, consequently, offspring fitness (Cohas et al. 2009; Harrison et al. 2011). If EP mating evolves as a mechanism to avoid inbreeding depression, a higher genetic relatedness between breeding females and their pair-bonded male would be expected in broods where females engage in EP mating. For example, in the red-backed fairy-wren (Malurus melanocephalus), females are more likely to engage in EP mating when they share a greater genetic similarity with their pair-bonded male (Varian-Ramos and Webster 2012). In addition to indirect genetic benefits, females might gain direct benefits from EP mating as a result of copulation with multiple males, including reduced harassment, access to additional resources, or securing a future partner (reviewed in Forstmeier et al. 2014). However, the main form of direct benefit that females might gain is increased parental care from additional males (Kempenaers 1993; Ihara 2002). This direct benefit, known as the “parental care” hypothesis, implies that females recruit extra males to help them in rearing the young, assuming that extra males provide care according to their certainty of paternity (e.g. Burke et al. 1989). Although females can potentially gain benefits provided by EP males, they may also risk the costly response of the cuckolded pair-bonded male. EP mating would decrease the genetic relatedness of the cuckolded pair-bonded males to the brood and, as such, these males are predicted to facultatively adjust their parental care in relation to their decreased paternity share in the brood (Sheldon 2002). These costs might constrain and influence the evolution of EP mating behavior. Cooperatively breeding species are excellent systems to examine female EP mating because the associated cost may be mitigated as a result of helpers’ efforts (Rubenstein 2007). In other words, if pair-bonded males decrease their “parental care” in response to female’s mating outside their pair bond (Albrecht et al. 2006), helpers might compensate for any deficits in parental care associated with less active pair-bonded males. For example, in the cooperatively breeding superb fairy-wren (Malurus cyaneus), though pair-bonded males reduce their parental care as a result of female EP mating, the contribution of helpers compensates for this lowered parental assistance, consequently liberating breeding females from constraints to seek additional EP matings (Mulder et al. 1994). Despite numerous investigations examining how female EP mating may influence a cuckolded male’s investment in broods, results to date have not been consistent across species. Some studies suggest that cuckolded males reduce parental investment in broods with EP mating, whereas others report no change or even an increase in parental care despite female EP mating (reviewed in Du et al. 2015). Moreover, in cooperatively breeding species, copulation outside the pair bond would alter the mean genetic relatedness of helpers to the resultant brood, particularly in species where helpers are related to the pair-bonded male. Thus, according to the kin selection theory (Hamilton 1964), related helpers are expected to reduce their efforts as a result of decreased relatedness, whereas unrelated helpers would be anticipated to not change their helping effort. Despite this, how helpers in cooperative systems respond to EP mating and how this is influenced by their genetic relatedness to the subsequent brood is poorly understood. Given this, we examine the responses of cuckolded males and helpers to EP mating in cooperatively breeding species where pair-bonded males provide a high level of paternal care, and both related and unrelated helpers actively provision broods. In this study, we first asked if EP mating functions as a mechanism for inbreeding avoidance and whether or not females gain “parental care” benefits in the cooperatively breeding noisy miner (Manorina melanocephala). Second, we examined how cuckolded pair-bonded males and male helpers of various relatedness levels respond to female EP mating behavior. The noisy miner is a honeyeater species from the Meliphagidae family endemic to wooded country in southeastern Australia. Noisy miners are a highly social species that lives year-round in large colonies characterized by a highly complex social structure. Most importantly for this study, noisy miners are cooperative breeders (Higgins et al. 2001), with both related and unrelated helpers contributing to the care of broods. Noisy miners are a particularly useful system in which to examine the influences of EP mating on the subsequent provisioning behavior of a cuckolded pair-bonded male for 2 reasons. First, helpers vary in the level of their genetic relatedness to both members of the breeding pair, with the breeding male providing the highest rate of brood provisioning (Barati 2017). Second, helpers are predominantly males with female helpers being rare, so there are minimal confounding effects of sex in helping (Barati 2017). Given this, we predict that 1) EP mating will be positively associated with the genetic similarity of pair-bonded mates (e.g. putative breeding pair); 2) if females seek extra parental care through EP mating, then EP males are expected to contribute more help to broods in which they have gained parentage compared to broods in which they have not; and 3) EP mating would result in reduction in the care provide by cuckolded males and related but not unrelated helpers as a consequence of their decreased genetic relatedness to the broods. METHODS Study populations and general fieldwork This study was conducted at 2 noisy miner colonies situated at Newholme Field Research Station (30°25’24”S, 151°38’84.38”E): a working rural property owned by the University of New England (UNE) and Dumaresq Dam Public Reserve, a public recreational site (30°30’S, 151°40’E). Both areas are located approximately 12 km northwest of Armidale, NSW, Australia. Between September 2013 and December 2015, noisy miners were captured and banded. Adult birds were caught with the aid of mist nets or baited walk-in cage traps before being fitted with a unique combination of 3 plastic color leg bands and 1 uniquely numbered metal band issued by the Australian Bird and Bat Banding Scheme. Birds were measured, aged as either less than or greater than 1 year of age and approximately 70 µL of blood was collected from the alar vein via venepuncture and placed in 70% ethanol and then stored at the Avian Behavioral Ecology Laboratory, UNE, at −3 °C for future analysis. Nestlings were banded and bled in the same manner at around 10–14 days post hatch. Nests were searched for every 2–3 days from mid-August each year and, once found, marked with a plastic numbered tag and visited at least every other day to examine their contents using a mirror attached to an extendable pole. Nest visits were made daily when nestlings were 13 days post hatch and close to fledging so that a precise fledge date could be noted for each brood. Observations of provisioning behavior From 13 September 2013 until 30 November 2015, we conducted behavioral observations at 29 nests in order to identify the putative breeding pair and helpers attending a given nest, whilst also collecting information on brood provisioning behavior. Observations were carried out from a hide placed 15–40 m (28.66 m ± 0.38 SE, N = 104) from nests. Observations lasted an average of 1 h in duration (57.88 min ± 1.83 SE). For each nest visit event, we dictated bird identity, as well as observation time, date, and location onto a digital recorder (Marantz PMD661, Japan) whilst viewing behaviors through either a telescope (Gerber Montana 15-45x) or binoculars (Monarch 7, 10x42, Nikon, Japan). Nest events were also recorded with a camera (2013–2014: analogue Hi8 camcorder, Sony, Japan; 2015: digital Panasonic HC-V270, Korea), placed 5.5 m ± 0.13 SE (N = 104) from the nest. Observations were carried out between 0800 and 1700 h. Molecular methods and genetic relatedness DNA was extracted in the Molecular Ecology Laboratory, UNE. Individuals were sexed using polymerase chain reactions (PCRs) (see DNA extraction methods and PCR condition in Supplementary Material) involving 1 primer pair (P2 and P8) simultaneously in order to amplify homologous parts of the CHD-W and CHD-Z genes (Griffiths et al. 1998). Birds were genotyped at 20 microsatellite loci previously isolated and characterized from noisy miners (Painter et al. 1997; Abbott et al. 2002; Kopps et al. 2013; Supplementary Table S1). The number of alleles per locus ranged from 3 to 15 (7.55 ± 0.64 SE, Supplementary Table S1). We tested our genotype data for deviations from Hardy–Weinberg equilibrium using CERVUS and found significant deviations in 6 of the 20 loci tested (Supplementary Table S1). Although these deviations do not impact our assessment of paternity because CERVUS simply excludes candidates based on mismatching genotypes, these deviations may be more problematic when inferring population-wide pairwise estimates of relatedness. Therefore, we excluded these 6 loci from subsequent analyses of pairwise genetic relatedness of female breeders with the pair-bonded male and their contingent of helpers. We determined pairwise genetic relatedness using the program KINGROUP v2 (Goodnight and Queller 1999; Konovalov et al. 2004). The relatedness coefficient is a measure of genetic similarity of 2 individuals based on the expected proportion of alleles that are identical by descent. These pairwise relatedness values range from −1 to +1, such that estimates close to zero represent the expected value for 2 randomly chosen individuals from the population, and increasingly positive values between 2 individuals indicate increasing levels of relatedness (Queller and Goodnight 1989). To test if pair-bonded male breeders and helpers were related or unrelated to breeding females at each nest, we performed a kinship test in KINGROUP v2, comparing the hypotheses that 2 individuals were related at the level of full siblings or parent-offspring (r = 0.5), or unrelated (r = 0), based on the likelihood ratio required to exclude 95% of 1000 simulated pairwise comparisons (Goodnight and Queller 1999). If neither hypothesis was significantly more likely than the other, r values were assumed to be approximately 0.25. Because these individuals were intermediate and not significantly different from unrelated (r = 0) and related (r = 0.5) individuals, they were placed into a third group: “unresolved” (McDonald et al. 2008a, b). Helpers and pair-bonded males were therefore placed into 3 groups with regards to their relatedness to breeding female: 1) birds that were significantly related (r = 0.5, N = 138); 2) birds that were significantly unrelated (r = 0, N = 24); and 3) birds with “unresolved” status (r = 0.25, N = 8) Parentage analysis and the identification of EP mating For each brood, the putative breeding female was identified based on the presence of strictly maternal behaviors such as nest construction, incubation of clutches and, if captured, the presence of a brood patch (Higgins et al. 2001; N = 8). The sex of putative females was then tested using the above molecular sexing method. Maternity was also confirmed with the parentage software CERVUS 3.0.7 using genotypes from 20 microsatellite loci (Marshall et al. 1998; Kalinowski et al. 2007). To determine paternity, we included all adult males at the colony in each breeding season as potential fathers. For each offspring, CERVUS calculates Trio LOD scores (natural logarithm of the likelihood ratio), giving the likelihood of paternity of that candidate parent relative to a randomly chosen individual in the population, while also considering known maternity. The differences in LOD scores (ΔLOD) between the 2 most likely fathers for a given offspring enabled the significance of the paternity assignment to be tested, with calibration according to simulations based on the parameters of the dataset. Candidate fathers suggested by CERVUS were then assigned to offspring if ΔLOD scores were ≥80% and there was not more than 1 allele mismatch. If there was more than 1 genetic father indicated for a given brood, and both males provisioned the focal brood (N = 2), we defined the pair-bonded male as the bird that exhibited a higher provisioning rate at the focal nest, given that breeding males typically provision more than helpers in this system (Barati et al., unpublished). Therefore, within each brood, the pair-bonded male was identified as the male that had paternity in the brood and provisioned at the highest rate among other males attending the nest. EP mating in a brood was assumed if 1) there was more than 1 sire in a brood (N = 3 broods) or 2) we could only identify a sire for some of the offspring in a brood but not all of them, suggesting that paternity was from unsampled males (N = 4 broods). We used the software COLONY 2.0 (Jones and Wang 2010) to quantify relatedness between siblings in a brood in the absence of known parents. By determining if individuals from the same brood were either full or half siblings, the absence or occurrence of EP mating could be detected. To test the validity of this method, we included both nestlings with known parents (N = 57) as determined with CERVUS and those broods with unsampled fathers (N = 18). Reconstructions of sibling relationships agreed with the CERVUS results, by probabilities of at least 90%. Therefore, when analyzing broods with unknown fathers, either half- or full-sibling relationships were assumed to be an accurate assignment if the probability produced by COLONY for that match was at least 0.9. Pair-bonded males were then categorized as significantly related or unrelated to the focal breeding female at a given nest using the same methodology as outlined above. Statistical analysis We used a goodness of fit test to determine if breeding males were more often unrelated to breeding females at their focal nest than expected by chance. The frequency distribution of different relatedness groups of helpers (sex and age), in relation to EP mating, was assessed with 2 × 2 contingency tables. To determine whether genetic relatedness between breeders and helpers differed with respect to EP mating, we constructed Generalized Linear Mixed Models (GLMMs), with EP status (binary variable with 2 groups) as a fixed effect and both nest identity and bird identity as separate random effects. In the model, genetic relatedness between breeders and helpers (continuous r values) were defined as the response variable. Similarly, to examine whether the number of helpers varied according to EP status, we used a GLMM with the number of helpers as a response variable, EP status as a fixed effect, and nest identity as a random effect. Further, to test whether EP status predicts the total provisioning rate, a GLMM was performed with EP status as a fixed effect and total provisioning rate as the response variable. In this model, we also included nest identity, bird identity, and observation order as random effects to control for nonindependence of data collected from multiple observations of the same individuals and same nests. In all GLMMs, the significance of each fixed effect was determined by comparing the fit of the model to that of the intercept-only model using likelihood ratio tests with an α = 0.05. Provisioning rate was the main index of parental behavior and helper effort in this breeding system, and increased with nestling age up to 11 days of age, after which it did not vary significantly (Barati 2017). Therefore, analyses examining provisioning rate only included observations collected 11–15 days post hatch. Previous analysis suggested that bird status was an important variable that influenced provisioning rate (Barati 2017). To examine if the presence of EP mating influenced provisioning rate beyond this, we first constructed a GLMM model with bird status (4 levels: breeder female, breeder male, related helper, and unrelated helper) as a fixed effect and then examined if adding EP mating (2 levels with binary distribution) improved model explanatory power. Similarly, to test if an interaction between status and EP mating improved model fit, we compared a model with status and EP mating with a model that had the same fixed effects plus their interactions. An interaction between “status” and EP mating would mean that birds with different “status” respond differently to EP mating and no interaction would suggest that birds with different “status” show similar responses to EP mating. Models were compared with likelihood ratio tests and the significance of terms was confirmed at α = 0.05. For each term, the effect size and 95% CIs are also reported. The response variable (provisioning rate) was square root transformed to normalize its distribution and to reduce residual variance. Models were run with a Gaussian distribution (link = “identity”). In addition, because female helpers were rare in this system, (~ 6% of helpers, N = 7) and were all unrelated to the breeding female at the nests provisioned, we only modeled male helper behavior to avoid the issue of rank deficiency. Similarly, within the group of “unsolved relatedness” helpers, none were present in groups with EP, therefore we excluded them (N = 3) from all analyses. All analyses were performed in the R statistical language and environment (R Core Team 2014). We used “lmer” function with REML approach in “lme4” package (Bates et al. 2014) to perform GLMM modeling. RESULTS Mate choice and genetic relatedness of breeders and male helpers In 86% of broods, females and pair-bonded males were classified as unrelated, with this proportion being significantly higher than that predicted by random mating ( χ12 = 7.1, P < 0.01). Mean relatedness between breeding females and the pair-bonded male breeders was significantly lower than the mean relatedness of breeding females with helper males provisioning at the same nest ( χ12 = 4.07, P = 0.04, β = 0.09, 95% CI = 0.002, 0.19; Figure 1). Figure 1 View largeDownload slide Mean ± SE genetic relatedness of breeding female to pair-bonded male and other males that provisioned her brood (e.g. helpers). Numbers below bars represent sample sizes for each group of birds. Figure 1 View largeDownload slide Mean ± SE genetic relatedness of breeding female to pair-bonded male and other males that provisioned her brood (e.g. helpers). Numbers below bars represent sample sizes for each group of birds. Genetic relatedness of breeders and helpers in relation to extra-pair mating Of the 75 offspring genotyped (N = 29 broods), we determined candidate fathers for 57 nestlings (N = 24 broods), with only 3 of these offspring (N = 3 broods) sired by a sampled male other than the pair-bonded male (2 with 0, and 1 with 1 allele mismatch). Seven of the 18 nestlings without a known sire occurred in broods (N = 7 broods) where we could identify fathers for some of the broods. Allele mismatches between these offspring and the most likely sampled sire ranged from 3 to 7 (3 mismatch: N = 2; 4 mismatch: N = 3; 6 mismatch: N = 1; and 7 mismatch: N = 1; mean ± SE: 4.42 ± 0.57). In some broods (N = 4) no known sire was determined for any of the nestlings. Subsequent analysis in COLONY showed that one nest contained a half-sibling to the other nestlings, suggesting an additional occurrence of EP mating. Therefore, we counted 11 nestlings (14% of all nestlings) across 8 broods (27% of all broods) that were sired by extra-pair males in these populations. There were either 1 (N = 5 broods) or 2 EP nestlings (N = 3 broods) per brood (mean ± SE: 1.37 ± 0.20), with all of these EP nests having broods of 3 offspring in total. The relatedness of the breeding pair did not differ between broods with and without EP mating ( χ12 = 0.03, P = 0.81, β = −0.01, 95% CI = −0.16, 0.12; Figure 2). The mean genetic relatedness of breeding females to male helpers did not vary in broods with and without EP mating ( χ12 = 1.16, P = 0.27, β = −0.06, 95% CI = −0.15, 0.03). Figure 2 View largeDownload slide Mean ± SE genetic relatedness of breeding female to the pair-bonded male and to male helpers in broods with extra-pair (EP) paternity and without EP paternity. Sample sizes (number of individuals in each group of birds) are shown below corresponding bar. Figure 2 View largeDownload slide Mean ± SE genetic relatedness of breeding female to the pair-bonded male and to male helpers in broods with extra-pair (EP) paternity and without EP paternity. Sample sizes (number of individuals in each group of birds) are shown below corresponding bar. Helper attendance and age/sex structure according to the presence of extra-pair mating Overall, we recorded 1,759 provisioning events in 21 broods without EP and 752 provisioning events in 8 broods with EP during a total of 112.28 h of nest observation across the 29 broods. During these, a total of 130 individuals provisioned focal broods. Brood size did not vary significantly between broods with and without EP occurrence ( χ12 = 0.3, P = 0.8). The mean number of helpers tended to be slightly greater in EP broods than in broods without EP; however, the differences were not significant ( χ12 = 1.7, P = 0.19, β = 1.4, 95% CI = −0.7, 3.71; Figure 3). The percentage of adult helpers (age ≥ 2) was significantly higher than that of first-year helpers in both broods with EP mating (74%, χ12 = 3.2, P < 0.05) and without EP mating (76%, χ12 = 9.03, P < 0.001). Figure 3 View largeDownload slide Mean ± SE number of helpers at nests with and without extra-pair (EP) paternity. Numbers below bars indicate sample sizes for each group of broods. Figure 3 View largeDownload slide Mean ± SE number of helpers at nests with and without extra-pair (EP) paternity. Numbers below bars indicate sample sizes for each group of broods. Broods with EP and without EP mating did not differ with regards to the helpers’ age structure (contingency table; χ12 = 0.004, P = 0.94). The sex ratio of helper contingents was extremely male-biased in both broods with EP and broods without EP (88% and 94%, respectively), and the frequency of male helpers also did not differ in relation to the occurrence of EP mating in a given nest (contingency table, χ12 = 0.27, P = 0.59). EP mating did not result in extra males attending the broods in 7 of the 8 EP broods (87%). The proportion of broods in which EP males did not attend was statistically higher than the proportion of broods that EP males provisioned ( χ12 = 10.11, P = 0.001). Provisioning response to extra-pair mating according to nest attendant status Overall provisioning rate was higher in broods in which EP mating was detected compared to those where it was not (Figure 3). As expected, a model including bird status (e.g. breeding female, pair-bonded male, and related and unrelated helpers) had significantly higher support in explaining variation in the provisioning rate of individuals when compared to an intercept-only model ( χ32 = 43.83, P < 0.001). Overall, breeding females provisioned broods at the highest rate followed by pair-bonded males, related, and then unrelated male helpers (Figure 4). A model with EP mating and bird status had significantly more support than a model with only bird status ( χ12 = 4.11, P = 0.04). However, adding the interaction of status and EP mating did not result in a significant difference in the variance explained ( χ32 = 0.44, P = 0.93), indicating that there was not a significant difference among birds of different status for their response to EP mating presence (Figure 4). Figure 4 View largeDownload slide Mean ± SE provisioning rate per hour by birds of different social class at the broods with and without extra-pair (EP) paternity. (BF: breeding female, BM: pair-bonded male, rH: related helper, and unrH: unrelated helper). Sample sizes for each category of birds are given below corresponding bar. Figure 4 View largeDownload slide Mean ± SE provisioning rate per hour by birds of different social class at the broods with and without extra-pair (EP) paternity. (BF: breeding female, BM: pair-bonded male, rH: related helper, and unrH: unrelated helper). Sample sizes for each category of birds are given below corresponding bar. DISCUSSION Frequency of extra-pair offspring in the noisy miner system Extra-pair males sired 14% of nestlings, whereas 27% of broods contained at least 1 EP nestling. These results are informative because previous observational data suggested that noisy miners were highly promiscuous (Dow 1978). However, the first molecular-based study found that EP mating was rare and only 3.5% of nestlings were sired by EP males (Poldmaa et al. 1995), a relatively low rate compared to other species typically considered promiscuous. For example, 85–90% of broods are reported to contain EP offspring in the superb fairy-wren (M. cyaneus; Mulder and Magrath 1994; Double and Cockburn 2000) and splendid fairy-wren (Malurus splendens; Brooker et al. 1990), whereas up to 80% of broods in the Australian magpie (Gymnorhina tibicen) are the result of EP mating (Durrant and Hughes 2005). In passerine bird species that exhibit EP mating, on average approximately 11% of offspring are found to be the result of EP paternity (Griffith et al. 2002). Therefore, the 14% EP offspring found in this study is close to the average EP mating rate for passerine bird species, seemingly contrasting the high level of promiscuous mating suggested by Dow (1978) prior to the advent of molecular techniques. The results herein differ from the level reported in the previous study (Poldmaa et al. 1995). The sample sizes used in the current study and Poldmaa et al. (1995) are similar (31 vs. 29 broods, respectively), so it is unlikely that the variation of the rate of EP mating found in this study and that of Poldmaa et al. (1995) stems from sample size variance. Differences in EP rates between these studies could be due to other factors such as colony structure and group composition of the focal study population. For example, it appears that relatedness within groups differed between these studies: Poldmaa et al. (1995) suggested that the monopolization of paternity by 1 male was a consequence of a high degree of genetic relatedness of helpers within each focal group. However, in the present study, helpers were not limited to related individuals and helper relatedness did not appear to drive EP patterns. It is possible that variation in EP levels between populations could be associated with different social environments, such as the composition of a given helper contingent, on a brood-by-brood basis. In addition, the differences between the EP rates found here and in Poldmaa et al. (1995), may also be a result of different methods used. Here, we used microsatellites marked for identifying parentage and therefore the rate of EP, whereas Poldmaa et al. (1995) used minisatellites. As these 2 methods are different in their resolution (Debrauwere et al. 1997), this may have impacted differences in observed estimation of EP rates. Inbreeding avoidance in noisy miners In the majority of broods, female and pair-bonded male breeders were not closely related. Despite the presence of both related and unrelated males in the helping group, females mated selectively with unrelated males more often than expected by chance, showing clear inbreeding avoidance. In most bird species, there is a general rule of avoiding inbreeding due to the fitness costs associated with mating with kin as a consequence of inbreeding depression (Frankham et al. 2002; Kokko and Ots 2006). For example, in purple-crowned fairy-wrens (Malurus coronatus), incestuous mating causes 30% hatching failure (Kingma et al. 2013). Therefore, various mechanisms have evolved for inbreeding avoidance in avian species. There are 2 possible mechanisms that seem most likely to allow female noisy miners to avoid mating with closely related individuals. First, although the sex ratio of offspring is not biased, there is a consistent male-biased adult sex ratio across populations as a consequence of female-biased dispersal and subsequent mortality (Barati et al., unpublished). This sex-biased dispersal naturally acts as a mechanism to reduce inbreeding, separating opposite-sex kin in space and therefore preventing matings between kin (Hazlitt et al. 2004, 2006; Guillaume and Perrin 2009; Liebgold et al. 2011). Dispersal acts as an important means of fostering inbreeding avoidance in other species as well, including the great tit (Parus major), where the level of inbreeding negatively correlated with dispersal distance from the natal breeding area (Szulkin and Sheldon 2008). Second, noisy miners might also use a form of kin recognition mechanism, which occurs in other cooperatively breeding birds (Jamieson et al. 2009) to avoid incestuous mating. This is particularly important for breeding females to avoid mating with the philopatric males that stay in the same territories and natal colony in this species. Females and males might use their complex acoustic repertoire systems (Holt et al. 2016) to differentiate between kin and nonkin when selecting mates. Noisy miners have previously shown the ability to differentiate between individuals using acoustic cues (McDonald 2012), and the closely related bell miner (Manorina melanophrys) uses acoustic cues to favor aiding kin (McDonald and Wright 2011), suggesting that a similar mechanism for inbreeding avoidance might be operating. Whether any discrimination occurs based on familiarity, an innate preference, or learnt template of a form of signal is currently unknown, but given that noisy miners also adjust helping effort towards relatives (Barati et al., unpublished), some form of kin recognition and thus avoidance during mating by breeding females seems highly likely. Although the genetic similarity of the pair-bonded breeding mate is assumed to be a driver of EP mating behavior, current evidence from avian systems is contradictory. Although genetic similarity between mates has shown to influence EP mating decisions by females (Kleven et al. 2005; Tarvin et al. 2005; Freeman-Gallant et al. 2006), in other studies a lack of relationship between mate genetic similarity and EP copulations has been reported (Kleven and Lifjeld 2005; Bouwman et al. 2006; Edly-Wright et al. 2007). One explanation is that the importance of EP mating for inbreeding avoidance is masked by other determinants such as dispersal patterns. EP mating is more likely to act as a mechanism for inbreeding avoidance in species that show a lack of dispersal. For example, incestuous mating in purple-crowned fairy-wrens (Malurus coronatus) occurs when sex-biased dispersal is limited (Kingma et al. 2009) and, as discussed above, the lack of juvenile dispersal can almost double the EP mating rate in Australian magpies (Durrant and Hughes 2005). The female-biased dispersal in noisy miners (Barati 2017) probably shapes inbreeding avoidance and differences in reliance upon dispersal patterns may well be the common factor in at least some of the contradictory results reported above. It is important, however, to note that although genetic similarity to the pair-bonded male does not appear to be a determinant of EP mating behavior in the noisy miner, females might still seek extra male copulations in order to benefit from their higher heterozygosity or genetic quality (Griffith et al. 2002; Harrison et al. 2013). This, however, needs further examinations in noisy miners. No evidence for extra-pair mating as a means by which females enhance help provided to broods Despite EP offspring being present in some broods, this did not result in additional care being provided by EP males. This is in contrast to the “communal polyandry” mating system for noisy miners suggested by Dow (1978) and further does not support the suggestion that female noisy miners engage in EP mating as a mechanism to recruit EP males as helpers (Dow 1978; Dow and Whitmore 1990). At the very least, if females are engaging in extra-pair matings, these are not resulting in high numbers of EP offspring. Furthermore, in some cooperatively breeding species, the number of helpers was found to be positively associated with EP mating. For example, in the superb starling (Lamprotornis superbus), the number of helpers within the group predicted the probability of EP mating behavior by breeding females. Females with a lower number of helpers were more likely to copulate with an EP male to gain the direct benefits of additional helpers (Rubenstein 2007). However, in the current study EP mating did not result in either a higher number of helpers or an increased rate of provisioning from successful EP males, indicating that the function of EP mating in noisy miners is unlikely to be related to the recruitment of extra helpers. Response of cuckolded males to female EP mating Overall, EP mating resulted in increased brood provisioning rates in EP broods compared to broods where the pair-bonded male obtained paternity of the entire brood. This increase in provision rates was the result of more helpers at EP nests. Contrary to predictions, cuckolded males did not reduce their provisioning rate towards broods where they had lost some share of paternity. Generally, the responses of cuckolded males to reduced paternity are thought to be influenced by 2 main determinants. First, a cuckolded male’s behavior towards a brood in which he has lost partial paternity should be a trade-off of the costs and benefits of continuing to care for that brood. Second, the abilities of male breeders to assess their share of parentage and the risk of cuckoldry are important in influencing their behaviors. In this study, we found that males suffering from reduced paternity through EP mating still succeeded at siring the majority of the brood with more than half of the offspring in a brood being sired by the pair-bonded male. This pattern of siring success has also been found in other species, such as the Mexican jay (Aphelocoma ultramarina), where cuckolded pair-bonded male breeders still provided the highest level of care to broods (Li and Brown 2002). One possibility is that females engaged in EP mating limit the parentage share of extra-pair males within broods to avoid cuckolded males reducing their level of care (Du et al. 2015). A recent meta-analysis across 48 species of fish, insects, birds, and mammals also demonstrated that the response of cuckolded males to female EP mating behavior depends on the strength of cuckoldry and the cost of parental care on the future reproductive success of cuckolded males (Griffin et al. 2013). Cuckolded males may therefore be flexible and relatively tolerant to female EP mating behavior if the parental care being provided does not negatively influence lifetime reproductive success (Grafen 1980). When examining the response of cuckolded males to a decreased share of paternity, it is important to note that a breeding male’s response is likely influenced by their ability to assess parentage share in the brood, which is typically thought to be difficult (Kempenaers and Sheldon 1997). Although cuckolded males can maximize their fitness by reducing parental care for unrelated offspring, low certainty about paternity would result in the risk of pair-bonded male breeders abandoning their own offspring (Maynard-Smith 1977; Wolf et al. 1988). Currently, the mechanisms of parentage detection are not well known in birds, and experimental manipulations of parentage have yielded inconsistent responses of cuckolded males (Kempenaers et al. 1998), suggesting that parentage assessment could be difficult and highly variable among species. In some species, the accessibility of females in their fertile period is assumed to act as a cue for males to assess their parentage (Davies et al. 1992; Komdeur 2001); however, this would be difficult to ascertain in a highly social species like the noisy miner. One possibility that cannot be ruled out in this study, given provisioning rates were only measured at the brood level rather than at the individual nestling level, is that cuckolded males only provisioned their own offspring when attending nests, ignoring any extra-pair nestlings. This would be possible if male breeders had some cues to identify extra-pair nestlings; however, preferential provision seems unlikely given the overall patterns of brood provisioning observed in this system. Nonetheless, this is an area worth examining with cross-fostering experiments that enable experimental manipulation of the paternity levels of breeding males. CONCLUSION In this study, we have shown that EP males sired 14% of offspring in the focal populations of noisy miner. This level of EP mating lies around the average rate for passerine birds, and is moderate in comparison to many other highly promiscuous species. This result, therefore, suggests that the mating system in noisy miner is unlikely to be highly promiscuous as proposed previously (Dow 1978; Brown 1987) but rather confirms a level more typical of passerines, but one that may be flexible from colony to colony. Two main hypotheses to explain female EP mating in birds suggest that this behavior might be beneficial in the form of avoiding inbreeding and through gaining extra parental care provided by EP males (Kempenaers and Dhondt 1993; Griffith et al. 2002). However, in the noisy miner cooperative breeding system, there was no evidence that EP mating might function as a mechanism to reduce the costs of inbreeding depression. We argue that other factors such as female-biased dispersal might function as a mechanism to avoid inbreeding, thus the relative importance of EP mating could be dependent on other ecological conditions. Further, EP mating did not either lead to additional help provided by EP males, or a reduction in the care provided by cuckolded males. Therefore, no evidence was found to support parental care hypothesis as a driver of EP mating in the noisy miner. Despite this, we cannot rule out that EP mating behavior may have other functions, such as increasing offspring fitness via increased heterozygosity or “genetic quality” (Griffith et al. 2002), or providing fertilization insurance in case the breeding male is infertile. SUPPLEMENTARY MATERIAL Supplementary data are available at Behavioral Ecology online. FUNDING The School of Environmental and Rural Sciences, UNE, provided financial support for this project. The project was also partly supported by the ANZ Equity Trustee Holsworth Wildlife Endowment grant. We are grateful to Farzaneh Etezadifar for her assistance during the fieldwork and Hugh Ford for suggestions on the initial draft of the manuscript. We thank 2 anonymous reviewers who provided constructive comments and helped to improve the earlier draft of this manuscript. We are grateful to Steve Debus and Heidi Kolkert who helped during some of the mist-netting and banding activities in 2013. This study was carried out in accordance with the approved (Protocol AEC13-142) guidelines and regulations of University of New England, Animal Ethics Committee. Data accessibility: Analyses reported in this article can be reproduced using the data provided by Barati et al. (2017). REFERENCES Abbott CL, Poldmaa T, Lougheed S, Clarke M, Boag PT. 2002. Hierarchical analysis of genetic population structure in the noisy miner using DNA microsatellite markers. Condor . 104: 652– 656. Google Scholar CrossRef Search ADS   Akçay E, Roughgarden J. 2007. Extra-pair paternity in birds: review of the genetic benefits. Evol Ecol Res . 9: 855– 868. Akçay Ç, Searcy WA, Campbell SE, Reed VA, Templeton CN. 2012. 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Extra-pair paternity is not driven by inbreeding avoidance and does not affect provisioning rates in a cooperatively breeding bird, the noisy miner (Manorina melanocephala)

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Abstract

Abstract In many socially monogamous bird species, both sexes regularly engage in mating outside their pair bond. Although the benefits of extra-pair (EP) mating behavior are clear and well established for males, such as an increase in the number of sired offspring, the benefits of EP mating behavior to females are less clear. A dominant theory for the incidence of EP mating is that socially monogamous females can improve the genetic quality of their offspring and avoid the costs of inbreeding through EP mating. In addition, in cooperatively breeding species, the theory of “parental care” predicts that females obtain additional help for their offspring through EP matings. Here, we examined evidence for both the inbreeding avoidance and parental care hypotheses in the cooperatively breeding noisy miner (Manorina melanocephala). Overall, EP mating occurred in 27% of broods, with 14% of offspring sired by males other than the identified pair-bonded male. There was a strong tendency to avoid pairing with genetically related individuals, with 86% of breeding pairs being significantly less related to each other than the general population. The occurrence of EP paternity was independent of the degree of relatedness between the pair. Provisioning patterns in relation to EP mating was not consistent with the “parental care” hypothesis and EP males did not contribute to the care of broods. These results demonstrate that in this system, there is no evidence that EP mating might function as a mechanism to reduce the costs of inbreeding depression or to gain benefits of extra helpers. INTRODUCTION Almost decades of developments in parentage analysis have confirmed a higher complexity and diversity of mating systems in birds than those predicted by social bonds alone, demonstrating that in most species females do not mate exclusively with their social partners, but rather also frequently copulate with males outside their social bond (Birkhead 1998). This phenomenon, referred to as extra-pair (EP) mating, is widespread among bird species. For example, only 10% of socially monogamous songbirds are found to be genetically monogamous (reviewed in Griffith et al. 2002). EP mating has broad implications and might influence mate choice as well as sexual conflicts and speciation (Bretman and Tregenza 2005). Although the mechanism and evolution of EP mating in birds have been the central focus of many recent studies, how EP mating evolves and what benefits might be accrued still remain a topic of considerable debate (reviewed in Griffith et al. 2002; Westneat and Stewart 2003; Akçay and Roughgarden 2007). Although EP mating enhances the reproductive opportunities for males, the benefits that females receive are less clear given that EP mating typically does not enhance fecundity (Møller and Birkhead 1994). Furthermore, mating with multiple males can impose significant costs to breeding females (Cornell and Tregenza 2007), such as aggression (Valera et al. 2003) or decreased parental care from her pair-bonded male (Arnqvist and Kirkpatrick 2005), as well as increased exposure to parasites and pathogens (Martinez-Padilla et al. 2012). Despite these costs, the near ubiquity of EP matings in the majority of taxa suggests that females should receive indirect and/or direct compensatory benefits to support this behavior, although the assumption that EP mating is adaptive might be incorrect under some circumstances (e.g. forced copulations controlled by males; Akçay et al. 2012). As a result, many hypotheses have been developed to explain what benefits females might gain for polyandrous mating behavior (Griffith et al. 2002). One of these hypotheses suggests that females use EP mating as a mechanism to avoid inbreeding and to gain an indirect benefit of improved offspring fitness (Griffith et al. 2002; Westneat and Stewart 2003; Akçay and Roughgarden 2007). For instance, inbreeding can cause negative effects on offspring traits such as survival or immunity (Keller and Waller 2002; Reid et al. 2011; Hemmings et al. 2012; Gohli et al. 2013) or even egg hatchability (Kingma et al. 2013). Therefore, EP mating with genetically dissimilar males might improve the genetic diversity of offspring (Kempenaers 2007) and, consequently, offspring fitness (Cohas et al. 2009; Harrison et al. 2011). If EP mating evolves as a mechanism to avoid inbreeding depression, a higher genetic relatedness between breeding females and their pair-bonded male would be expected in broods where females engage in EP mating. For example, in the red-backed fairy-wren (Malurus melanocephalus), females are more likely to engage in EP mating when they share a greater genetic similarity with their pair-bonded male (Varian-Ramos and Webster 2012). In addition to indirect genetic benefits, females might gain direct benefits from EP mating as a result of copulation with multiple males, including reduced harassment, access to additional resources, or securing a future partner (reviewed in Forstmeier et al. 2014). However, the main form of direct benefit that females might gain is increased parental care from additional males (Kempenaers 1993; Ihara 2002). This direct benefit, known as the “parental care” hypothesis, implies that females recruit extra males to help them in rearing the young, assuming that extra males provide care according to their certainty of paternity (e.g. Burke et al. 1989). Although females can potentially gain benefits provided by EP males, they may also risk the costly response of the cuckolded pair-bonded male. EP mating would decrease the genetic relatedness of the cuckolded pair-bonded males to the brood and, as such, these males are predicted to facultatively adjust their parental care in relation to their decreased paternity share in the brood (Sheldon 2002). These costs might constrain and influence the evolution of EP mating behavior. Cooperatively breeding species are excellent systems to examine female EP mating because the associated cost may be mitigated as a result of helpers’ efforts (Rubenstein 2007). In other words, if pair-bonded males decrease their “parental care” in response to female’s mating outside their pair bond (Albrecht et al. 2006), helpers might compensate for any deficits in parental care associated with less active pair-bonded males. For example, in the cooperatively breeding superb fairy-wren (Malurus cyaneus), though pair-bonded males reduce their parental care as a result of female EP mating, the contribution of helpers compensates for this lowered parental assistance, consequently liberating breeding females from constraints to seek additional EP matings (Mulder et al. 1994). Despite numerous investigations examining how female EP mating may influence a cuckolded male’s investment in broods, results to date have not been consistent across species. Some studies suggest that cuckolded males reduce parental investment in broods with EP mating, whereas others report no change or even an increase in parental care despite female EP mating (reviewed in Du et al. 2015). Moreover, in cooperatively breeding species, copulation outside the pair bond would alter the mean genetic relatedness of helpers to the resultant brood, particularly in species where helpers are related to the pair-bonded male. Thus, according to the kin selection theory (Hamilton 1964), related helpers are expected to reduce their efforts as a result of decreased relatedness, whereas unrelated helpers would be anticipated to not change their helping effort. Despite this, how helpers in cooperative systems respond to EP mating and how this is influenced by their genetic relatedness to the subsequent brood is poorly understood. Given this, we examine the responses of cuckolded males and helpers to EP mating in cooperatively breeding species where pair-bonded males provide a high level of paternal care, and both related and unrelated helpers actively provision broods. In this study, we first asked if EP mating functions as a mechanism for inbreeding avoidance and whether or not females gain “parental care” benefits in the cooperatively breeding noisy miner (Manorina melanocephala). Second, we examined how cuckolded pair-bonded males and male helpers of various relatedness levels respond to female EP mating behavior. The noisy miner is a honeyeater species from the Meliphagidae family endemic to wooded country in southeastern Australia. Noisy miners are a highly social species that lives year-round in large colonies characterized by a highly complex social structure. Most importantly for this study, noisy miners are cooperative breeders (Higgins et al. 2001), with both related and unrelated helpers contributing to the care of broods. Noisy miners are a particularly useful system in which to examine the influences of EP mating on the subsequent provisioning behavior of a cuckolded pair-bonded male for 2 reasons. First, helpers vary in the level of their genetic relatedness to both members of the breeding pair, with the breeding male providing the highest rate of brood provisioning (Barati 2017). Second, helpers are predominantly males with female helpers being rare, so there are minimal confounding effects of sex in helping (Barati 2017). Given this, we predict that 1) EP mating will be positively associated with the genetic similarity of pair-bonded mates (e.g. putative breeding pair); 2) if females seek extra parental care through EP mating, then EP males are expected to contribute more help to broods in which they have gained parentage compared to broods in which they have not; and 3) EP mating would result in reduction in the care provide by cuckolded males and related but not unrelated helpers as a consequence of their decreased genetic relatedness to the broods. METHODS Study populations and general fieldwork This study was conducted at 2 noisy miner colonies situated at Newholme Field Research Station (30°25’24”S, 151°38’84.38”E): a working rural property owned by the University of New England (UNE) and Dumaresq Dam Public Reserve, a public recreational site (30°30’S, 151°40’E). Both areas are located approximately 12 km northwest of Armidale, NSW, Australia. Between September 2013 and December 2015, noisy miners were captured and banded. Adult birds were caught with the aid of mist nets or baited walk-in cage traps before being fitted with a unique combination of 3 plastic color leg bands and 1 uniquely numbered metal band issued by the Australian Bird and Bat Banding Scheme. Birds were measured, aged as either less than or greater than 1 year of age and approximately 70 µL of blood was collected from the alar vein via venepuncture and placed in 70% ethanol and then stored at the Avian Behavioral Ecology Laboratory, UNE, at −3 °C for future analysis. Nestlings were banded and bled in the same manner at around 10–14 days post hatch. Nests were searched for every 2–3 days from mid-August each year and, once found, marked with a plastic numbered tag and visited at least every other day to examine their contents using a mirror attached to an extendable pole. Nest visits were made daily when nestlings were 13 days post hatch and close to fledging so that a precise fledge date could be noted for each brood. Observations of provisioning behavior From 13 September 2013 until 30 November 2015, we conducted behavioral observations at 29 nests in order to identify the putative breeding pair and helpers attending a given nest, whilst also collecting information on brood provisioning behavior. Observations were carried out from a hide placed 15–40 m (28.66 m ± 0.38 SE, N = 104) from nests. Observations lasted an average of 1 h in duration (57.88 min ± 1.83 SE). For each nest visit event, we dictated bird identity, as well as observation time, date, and location onto a digital recorder (Marantz PMD661, Japan) whilst viewing behaviors through either a telescope (Gerber Montana 15-45x) or binoculars (Monarch 7, 10x42, Nikon, Japan). Nest events were also recorded with a camera (2013–2014: analogue Hi8 camcorder, Sony, Japan; 2015: digital Panasonic HC-V270, Korea), placed 5.5 m ± 0.13 SE (N = 104) from the nest. Observations were carried out between 0800 and 1700 h. Molecular methods and genetic relatedness DNA was extracted in the Molecular Ecology Laboratory, UNE. Individuals were sexed using polymerase chain reactions (PCRs) (see DNA extraction methods and PCR condition in Supplementary Material) involving 1 primer pair (P2 and P8) simultaneously in order to amplify homologous parts of the CHD-W and CHD-Z genes (Griffiths et al. 1998). Birds were genotyped at 20 microsatellite loci previously isolated and characterized from noisy miners (Painter et al. 1997; Abbott et al. 2002; Kopps et al. 2013; Supplementary Table S1). The number of alleles per locus ranged from 3 to 15 (7.55 ± 0.64 SE, Supplementary Table S1). We tested our genotype data for deviations from Hardy–Weinberg equilibrium using CERVUS and found significant deviations in 6 of the 20 loci tested (Supplementary Table S1). Although these deviations do not impact our assessment of paternity because CERVUS simply excludes candidates based on mismatching genotypes, these deviations may be more problematic when inferring population-wide pairwise estimates of relatedness. Therefore, we excluded these 6 loci from subsequent analyses of pairwise genetic relatedness of female breeders with the pair-bonded male and their contingent of helpers. We determined pairwise genetic relatedness using the program KINGROUP v2 (Goodnight and Queller 1999; Konovalov et al. 2004). The relatedness coefficient is a measure of genetic similarity of 2 individuals based on the expected proportion of alleles that are identical by descent. These pairwise relatedness values range from −1 to +1, such that estimates close to zero represent the expected value for 2 randomly chosen individuals from the population, and increasingly positive values between 2 individuals indicate increasing levels of relatedness (Queller and Goodnight 1989). To test if pair-bonded male breeders and helpers were related or unrelated to breeding females at each nest, we performed a kinship test in KINGROUP v2, comparing the hypotheses that 2 individuals were related at the level of full siblings or parent-offspring (r = 0.5), or unrelated (r = 0), based on the likelihood ratio required to exclude 95% of 1000 simulated pairwise comparisons (Goodnight and Queller 1999). If neither hypothesis was significantly more likely than the other, r values were assumed to be approximately 0.25. Because these individuals were intermediate and not significantly different from unrelated (r = 0) and related (r = 0.5) individuals, they were placed into a third group: “unresolved” (McDonald et al. 2008a, b). Helpers and pair-bonded males were therefore placed into 3 groups with regards to their relatedness to breeding female: 1) birds that were significantly related (r = 0.5, N = 138); 2) birds that were significantly unrelated (r = 0, N = 24); and 3) birds with “unresolved” status (r = 0.25, N = 8) Parentage analysis and the identification of EP mating For each brood, the putative breeding female was identified based on the presence of strictly maternal behaviors such as nest construction, incubation of clutches and, if captured, the presence of a brood patch (Higgins et al. 2001; N = 8). The sex of putative females was then tested using the above molecular sexing method. Maternity was also confirmed with the parentage software CERVUS 3.0.7 using genotypes from 20 microsatellite loci (Marshall et al. 1998; Kalinowski et al. 2007). To determine paternity, we included all adult males at the colony in each breeding season as potential fathers. For each offspring, CERVUS calculates Trio LOD scores (natural logarithm of the likelihood ratio), giving the likelihood of paternity of that candidate parent relative to a randomly chosen individual in the population, while also considering known maternity. The differences in LOD scores (ΔLOD) between the 2 most likely fathers for a given offspring enabled the significance of the paternity assignment to be tested, with calibration according to simulations based on the parameters of the dataset. Candidate fathers suggested by CERVUS were then assigned to offspring if ΔLOD scores were ≥80% and there was not more than 1 allele mismatch. If there was more than 1 genetic father indicated for a given brood, and both males provisioned the focal brood (N = 2), we defined the pair-bonded male as the bird that exhibited a higher provisioning rate at the focal nest, given that breeding males typically provision more than helpers in this system (Barati et al., unpublished). Therefore, within each brood, the pair-bonded male was identified as the male that had paternity in the brood and provisioned at the highest rate among other males attending the nest. EP mating in a brood was assumed if 1) there was more than 1 sire in a brood (N = 3 broods) or 2) we could only identify a sire for some of the offspring in a brood but not all of them, suggesting that paternity was from unsampled males (N = 4 broods). We used the software COLONY 2.0 (Jones and Wang 2010) to quantify relatedness between siblings in a brood in the absence of known parents. By determining if individuals from the same brood were either full or half siblings, the absence or occurrence of EP mating could be detected. To test the validity of this method, we included both nestlings with known parents (N = 57) as determined with CERVUS and those broods with unsampled fathers (N = 18). Reconstructions of sibling relationships agreed with the CERVUS results, by probabilities of at least 90%. Therefore, when analyzing broods with unknown fathers, either half- or full-sibling relationships were assumed to be an accurate assignment if the probability produced by COLONY for that match was at least 0.9. Pair-bonded males were then categorized as significantly related or unrelated to the focal breeding female at a given nest using the same methodology as outlined above. Statistical analysis We used a goodness of fit test to determine if breeding males were more often unrelated to breeding females at their focal nest than expected by chance. The frequency distribution of different relatedness groups of helpers (sex and age), in relation to EP mating, was assessed with 2 × 2 contingency tables. To determine whether genetic relatedness between breeders and helpers differed with respect to EP mating, we constructed Generalized Linear Mixed Models (GLMMs), with EP status (binary variable with 2 groups) as a fixed effect and both nest identity and bird identity as separate random effects. In the model, genetic relatedness between breeders and helpers (continuous r values) were defined as the response variable. Similarly, to examine whether the number of helpers varied according to EP status, we used a GLMM with the number of helpers as a response variable, EP status as a fixed effect, and nest identity as a random effect. Further, to test whether EP status predicts the total provisioning rate, a GLMM was performed with EP status as a fixed effect and total provisioning rate as the response variable. In this model, we also included nest identity, bird identity, and observation order as random effects to control for nonindependence of data collected from multiple observations of the same individuals and same nests. In all GLMMs, the significance of each fixed effect was determined by comparing the fit of the model to that of the intercept-only model using likelihood ratio tests with an α = 0.05. Provisioning rate was the main index of parental behavior and helper effort in this breeding system, and increased with nestling age up to 11 days of age, after which it did not vary significantly (Barati 2017). Therefore, analyses examining provisioning rate only included observations collected 11–15 days post hatch. Previous analysis suggested that bird status was an important variable that influenced provisioning rate (Barati 2017). To examine if the presence of EP mating influenced provisioning rate beyond this, we first constructed a GLMM model with bird status (4 levels: breeder female, breeder male, related helper, and unrelated helper) as a fixed effect and then examined if adding EP mating (2 levels with binary distribution) improved model explanatory power. Similarly, to test if an interaction between status and EP mating improved model fit, we compared a model with status and EP mating with a model that had the same fixed effects plus their interactions. An interaction between “status” and EP mating would mean that birds with different “status” respond differently to EP mating and no interaction would suggest that birds with different “status” show similar responses to EP mating. Models were compared with likelihood ratio tests and the significance of terms was confirmed at α = 0.05. For each term, the effect size and 95% CIs are also reported. The response variable (provisioning rate) was square root transformed to normalize its distribution and to reduce residual variance. Models were run with a Gaussian distribution (link = “identity”). In addition, because female helpers were rare in this system, (~ 6% of helpers, N = 7) and were all unrelated to the breeding female at the nests provisioned, we only modeled male helper behavior to avoid the issue of rank deficiency. Similarly, within the group of “unsolved relatedness” helpers, none were present in groups with EP, therefore we excluded them (N = 3) from all analyses. All analyses were performed in the R statistical language and environment (R Core Team 2014). We used “lmer” function with REML approach in “lme4” package (Bates et al. 2014) to perform GLMM modeling. RESULTS Mate choice and genetic relatedness of breeders and male helpers In 86% of broods, females and pair-bonded males were classified as unrelated, with this proportion being significantly higher than that predicted by random mating ( χ12 = 7.1, P < 0.01). Mean relatedness between breeding females and the pair-bonded male breeders was significantly lower than the mean relatedness of breeding females with helper males provisioning at the same nest ( χ12 = 4.07, P = 0.04, β = 0.09, 95% CI = 0.002, 0.19; Figure 1). Figure 1 View largeDownload slide Mean ± SE genetic relatedness of breeding female to pair-bonded male and other males that provisioned her brood (e.g. helpers). Numbers below bars represent sample sizes for each group of birds. Figure 1 View largeDownload slide Mean ± SE genetic relatedness of breeding female to pair-bonded male and other males that provisioned her brood (e.g. helpers). Numbers below bars represent sample sizes for each group of birds. Genetic relatedness of breeders and helpers in relation to extra-pair mating Of the 75 offspring genotyped (N = 29 broods), we determined candidate fathers for 57 nestlings (N = 24 broods), with only 3 of these offspring (N = 3 broods) sired by a sampled male other than the pair-bonded male (2 with 0, and 1 with 1 allele mismatch). Seven of the 18 nestlings without a known sire occurred in broods (N = 7 broods) where we could identify fathers for some of the broods. Allele mismatches between these offspring and the most likely sampled sire ranged from 3 to 7 (3 mismatch: N = 2; 4 mismatch: N = 3; 6 mismatch: N = 1; and 7 mismatch: N = 1; mean ± SE: 4.42 ± 0.57). In some broods (N = 4) no known sire was determined for any of the nestlings. Subsequent analysis in COLONY showed that one nest contained a half-sibling to the other nestlings, suggesting an additional occurrence of EP mating. Therefore, we counted 11 nestlings (14% of all nestlings) across 8 broods (27% of all broods) that were sired by extra-pair males in these populations. There were either 1 (N = 5 broods) or 2 EP nestlings (N = 3 broods) per brood (mean ± SE: 1.37 ± 0.20), with all of these EP nests having broods of 3 offspring in total. The relatedness of the breeding pair did not differ between broods with and without EP mating ( χ12 = 0.03, P = 0.81, β = −0.01, 95% CI = −0.16, 0.12; Figure 2). The mean genetic relatedness of breeding females to male helpers did not vary in broods with and without EP mating ( χ12 = 1.16, P = 0.27, β = −0.06, 95% CI = −0.15, 0.03). Figure 2 View largeDownload slide Mean ± SE genetic relatedness of breeding female to the pair-bonded male and to male helpers in broods with extra-pair (EP) paternity and without EP paternity. Sample sizes (number of individuals in each group of birds) are shown below corresponding bar. Figure 2 View largeDownload slide Mean ± SE genetic relatedness of breeding female to the pair-bonded male and to male helpers in broods with extra-pair (EP) paternity and without EP paternity. Sample sizes (number of individuals in each group of birds) are shown below corresponding bar. Helper attendance and age/sex structure according to the presence of extra-pair mating Overall, we recorded 1,759 provisioning events in 21 broods without EP and 752 provisioning events in 8 broods with EP during a total of 112.28 h of nest observation across the 29 broods. During these, a total of 130 individuals provisioned focal broods. Brood size did not vary significantly between broods with and without EP occurrence ( χ12 = 0.3, P = 0.8). The mean number of helpers tended to be slightly greater in EP broods than in broods without EP; however, the differences were not significant ( χ12 = 1.7, P = 0.19, β = 1.4, 95% CI = −0.7, 3.71; Figure 3). The percentage of adult helpers (age ≥ 2) was significantly higher than that of first-year helpers in both broods with EP mating (74%, χ12 = 3.2, P < 0.05) and without EP mating (76%, χ12 = 9.03, P < 0.001). Figure 3 View largeDownload slide Mean ± SE number of helpers at nests with and without extra-pair (EP) paternity. Numbers below bars indicate sample sizes for each group of broods. Figure 3 View largeDownload slide Mean ± SE number of helpers at nests with and without extra-pair (EP) paternity. Numbers below bars indicate sample sizes for each group of broods. Broods with EP and without EP mating did not differ with regards to the helpers’ age structure (contingency table; χ12 = 0.004, P = 0.94). The sex ratio of helper contingents was extremely male-biased in both broods with EP and broods without EP (88% and 94%, respectively), and the frequency of male helpers also did not differ in relation to the occurrence of EP mating in a given nest (contingency table, χ12 = 0.27, P = 0.59). EP mating did not result in extra males attending the broods in 7 of the 8 EP broods (87%). The proportion of broods in which EP males did not attend was statistically higher than the proportion of broods that EP males provisioned ( χ12 = 10.11, P = 0.001). Provisioning response to extra-pair mating according to nest attendant status Overall provisioning rate was higher in broods in which EP mating was detected compared to those where it was not (Figure 3). As expected, a model including bird status (e.g. breeding female, pair-bonded male, and related and unrelated helpers) had significantly higher support in explaining variation in the provisioning rate of individuals when compared to an intercept-only model ( χ32 = 43.83, P < 0.001). Overall, breeding females provisioned broods at the highest rate followed by pair-bonded males, related, and then unrelated male helpers (Figure 4). A model with EP mating and bird status had significantly more support than a model with only bird status ( χ12 = 4.11, P = 0.04). However, adding the interaction of status and EP mating did not result in a significant difference in the variance explained ( χ32 = 0.44, P = 0.93), indicating that there was not a significant difference among birds of different status for their response to EP mating presence (Figure 4). Figure 4 View largeDownload slide Mean ± SE provisioning rate per hour by birds of different social class at the broods with and without extra-pair (EP) paternity. (BF: breeding female, BM: pair-bonded male, rH: related helper, and unrH: unrelated helper). Sample sizes for each category of birds are given below corresponding bar. Figure 4 View largeDownload slide Mean ± SE provisioning rate per hour by birds of different social class at the broods with and without extra-pair (EP) paternity. (BF: breeding female, BM: pair-bonded male, rH: related helper, and unrH: unrelated helper). Sample sizes for each category of birds are given below corresponding bar. DISCUSSION Frequency of extra-pair offspring in the noisy miner system Extra-pair males sired 14% of nestlings, whereas 27% of broods contained at least 1 EP nestling. These results are informative because previous observational data suggested that noisy miners were highly promiscuous (Dow 1978). However, the first molecular-based study found that EP mating was rare and only 3.5% of nestlings were sired by EP males (Poldmaa et al. 1995), a relatively low rate compared to other species typically considered promiscuous. For example, 85–90% of broods are reported to contain EP offspring in the superb fairy-wren (M. cyaneus; Mulder and Magrath 1994; Double and Cockburn 2000) and splendid fairy-wren (Malurus splendens; Brooker et al. 1990), whereas up to 80% of broods in the Australian magpie (Gymnorhina tibicen) are the result of EP mating (Durrant and Hughes 2005). In passerine bird species that exhibit EP mating, on average approximately 11% of offspring are found to be the result of EP paternity (Griffith et al. 2002). Therefore, the 14% EP offspring found in this study is close to the average EP mating rate for passerine bird species, seemingly contrasting the high level of promiscuous mating suggested by Dow (1978) prior to the advent of molecular techniques. The results herein differ from the level reported in the previous study (Poldmaa et al. 1995). The sample sizes used in the current study and Poldmaa et al. (1995) are similar (31 vs. 29 broods, respectively), so it is unlikely that the variation of the rate of EP mating found in this study and that of Poldmaa et al. (1995) stems from sample size variance. Differences in EP rates between these studies could be due to other factors such as colony structure and group composition of the focal study population. For example, it appears that relatedness within groups differed between these studies: Poldmaa et al. (1995) suggested that the monopolization of paternity by 1 male was a consequence of a high degree of genetic relatedness of helpers within each focal group. However, in the present study, helpers were not limited to related individuals and helper relatedness did not appear to drive EP patterns. It is possible that variation in EP levels between populations could be associated with different social environments, such as the composition of a given helper contingent, on a brood-by-brood basis. In addition, the differences between the EP rates found here and in Poldmaa et al. (1995), may also be a result of different methods used. Here, we used microsatellites marked for identifying parentage and therefore the rate of EP, whereas Poldmaa et al. (1995) used minisatellites. As these 2 methods are different in their resolution (Debrauwere et al. 1997), this may have impacted differences in observed estimation of EP rates. Inbreeding avoidance in noisy miners In the majority of broods, female and pair-bonded male breeders were not closely related. Despite the presence of both related and unrelated males in the helping group, females mated selectively with unrelated males more often than expected by chance, showing clear inbreeding avoidance. In most bird species, there is a general rule of avoiding inbreeding due to the fitness costs associated with mating with kin as a consequence of inbreeding depression (Frankham et al. 2002; Kokko and Ots 2006). For example, in purple-crowned fairy-wrens (Malurus coronatus), incestuous mating causes 30% hatching failure (Kingma et al. 2013). Therefore, various mechanisms have evolved for inbreeding avoidance in avian species. There are 2 possible mechanisms that seem most likely to allow female noisy miners to avoid mating with closely related individuals. First, although the sex ratio of offspring is not biased, there is a consistent male-biased adult sex ratio across populations as a consequence of female-biased dispersal and subsequent mortality (Barati et al., unpublished). This sex-biased dispersal naturally acts as a mechanism to reduce inbreeding, separating opposite-sex kin in space and therefore preventing matings between kin (Hazlitt et al. 2004, 2006; Guillaume and Perrin 2009; Liebgold et al. 2011). Dispersal acts as an important means of fostering inbreeding avoidance in other species as well, including the great tit (Parus major), where the level of inbreeding negatively correlated with dispersal distance from the natal breeding area (Szulkin and Sheldon 2008). Second, noisy miners might also use a form of kin recognition mechanism, which occurs in other cooperatively breeding birds (Jamieson et al. 2009) to avoid incestuous mating. This is particularly important for breeding females to avoid mating with the philopatric males that stay in the same territories and natal colony in this species. Females and males might use their complex acoustic repertoire systems (Holt et al. 2016) to differentiate between kin and nonkin when selecting mates. Noisy miners have previously shown the ability to differentiate between individuals using acoustic cues (McDonald 2012), and the closely related bell miner (Manorina melanophrys) uses acoustic cues to favor aiding kin (McDonald and Wright 2011), suggesting that a similar mechanism for inbreeding avoidance might be operating. Whether any discrimination occurs based on familiarity, an innate preference, or learnt template of a form of signal is currently unknown, but given that noisy miners also adjust helping effort towards relatives (Barati et al., unpublished), some form of kin recognition and thus avoidance during mating by breeding females seems highly likely. Although the genetic similarity of the pair-bonded breeding mate is assumed to be a driver of EP mating behavior, current evidence from avian systems is contradictory. Although genetic similarity between mates has shown to influence EP mating decisions by females (Kleven et al. 2005; Tarvin et al. 2005; Freeman-Gallant et al. 2006), in other studies a lack of relationship between mate genetic similarity and EP copulations has been reported (Kleven and Lifjeld 2005; Bouwman et al. 2006; Edly-Wright et al. 2007). One explanation is that the importance of EP mating for inbreeding avoidance is masked by other determinants such as dispersal patterns. EP mating is more likely to act as a mechanism for inbreeding avoidance in species that show a lack of dispersal. For example, incestuous mating in purple-crowned fairy-wrens (Malurus coronatus) occurs when sex-biased dispersal is limited (Kingma et al. 2009) and, as discussed above, the lack of juvenile dispersal can almost double the EP mating rate in Australian magpies (Durrant and Hughes 2005). The female-biased dispersal in noisy miners (Barati 2017) probably shapes inbreeding avoidance and differences in reliance upon dispersal patterns may well be the common factor in at least some of the contradictory results reported above. It is important, however, to note that although genetic similarity to the pair-bonded male does not appear to be a determinant of EP mating behavior in the noisy miner, females might still seek extra male copulations in order to benefit from their higher heterozygosity or genetic quality (Griffith et al. 2002; Harrison et al. 2013). This, however, needs further examinations in noisy miners. No evidence for extra-pair mating as a means by which females enhance help provided to broods Despite EP offspring being present in some broods, this did not result in additional care being provided by EP males. This is in contrast to the “communal polyandry” mating system for noisy miners suggested by Dow (1978) and further does not support the suggestion that female noisy miners engage in EP mating as a mechanism to recruit EP males as helpers (Dow 1978; Dow and Whitmore 1990). At the very least, if females are engaging in extra-pair matings, these are not resulting in high numbers of EP offspring. Furthermore, in some cooperatively breeding species, the number of helpers was found to be positively associated with EP mating. For example, in the superb starling (Lamprotornis superbus), the number of helpers within the group predicted the probability of EP mating behavior by breeding females. Females with a lower number of helpers were more likely to copulate with an EP male to gain the direct benefits of additional helpers (Rubenstein 2007). However, in the current study EP mating did not result in either a higher number of helpers or an increased rate of provisioning from successful EP males, indicating that the function of EP mating in noisy miners is unlikely to be related to the recruitment of extra helpers. Response of cuckolded males to female EP mating Overall, EP mating resulted in increased brood provisioning rates in EP broods compared to broods where the pair-bonded male obtained paternity of the entire brood. This increase in provision rates was the result of more helpers at EP nests. Contrary to predictions, cuckolded males did not reduce their provisioning rate towards broods where they had lost some share of paternity. Generally, the responses of cuckolded males to reduced paternity are thought to be influenced by 2 main determinants. First, a cuckolded male’s behavior towards a brood in which he has lost partial paternity should be a trade-off of the costs and benefits of continuing to care for that brood. Second, the abilities of male breeders to assess their share of parentage and the risk of cuckoldry are important in influencing their behaviors. In this study, we found that males suffering from reduced paternity through EP mating still succeeded at siring the majority of the brood with more than half of the offspring in a brood being sired by the pair-bonded male. This pattern of siring success has also been found in other species, such as the Mexican jay (Aphelocoma ultramarina), where cuckolded pair-bonded male breeders still provided the highest level of care to broods (Li and Brown 2002). One possibility is that females engaged in EP mating limit the parentage share of extra-pair males within broods to avoid cuckolded males reducing their level of care (Du et al. 2015). A recent meta-analysis across 48 species of fish, insects, birds, and mammals also demonstrated that the response of cuckolded males to female EP mating behavior depends on the strength of cuckoldry and the cost of parental care on the future reproductive success of cuckolded males (Griffin et al. 2013). Cuckolded males may therefore be flexible and relatively tolerant to female EP mating behavior if the parental care being provided does not negatively influence lifetime reproductive success (Grafen 1980). When examining the response of cuckolded males to a decreased share of paternity, it is important to note that a breeding male’s response is likely influenced by their ability to assess parentage share in the brood, which is typically thought to be difficult (Kempenaers and Sheldon 1997). Although cuckolded males can maximize their fitness by reducing parental care for unrelated offspring, low certainty about paternity would result in the risk of pair-bonded male breeders abandoning their own offspring (Maynard-Smith 1977; Wolf et al. 1988). Currently, the mechanisms of parentage detection are not well known in birds, and experimental manipulations of parentage have yielded inconsistent responses of cuckolded males (Kempenaers et al. 1998), suggesting that parentage assessment could be difficult and highly variable among species. In some species, the accessibility of females in their fertile period is assumed to act as a cue for males to assess their parentage (Davies et al. 1992; Komdeur 2001); however, this would be difficult to ascertain in a highly social species like the noisy miner. One possibility that cannot be ruled out in this study, given provisioning rates were only measured at the brood level rather than at the individual nestling level, is that cuckolded males only provisioned their own offspring when attending nests, ignoring any extra-pair nestlings. This would be possible if male breeders had some cues to identify extra-pair nestlings; however, preferential provision seems unlikely given the overall patterns of brood provisioning observed in this system. Nonetheless, this is an area worth examining with cross-fostering experiments that enable experimental manipulation of the paternity levels of breeding males. CONCLUSION In this study, we have shown that EP males sired 14% of offspring in the focal populations of noisy miner. This level of EP mating lies around the average rate for passerine birds, and is moderate in comparison to many other highly promiscuous species. This result, therefore, suggests that the mating system in noisy miner is unlikely to be highly promiscuous as proposed previously (Dow 1978; Brown 1987) but rather confirms a level more typical of passerines, but one that may be flexible from colony to colony. Two main hypotheses to explain female EP mating in birds suggest that this behavior might be beneficial in the form of avoiding inbreeding and through gaining extra parental care provided by EP males (Kempenaers and Dhondt 1993; Griffith et al. 2002). However, in the noisy miner cooperative breeding system, there was no evidence that EP mating might function as a mechanism to reduce the costs of inbreeding depression. We argue that other factors such as female-biased dispersal might function as a mechanism to avoid inbreeding, thus the relative importance of EP mating could be dependent on other ecological conditions. Further, EP mating did not either lead to additional help provided by EP males, or a reduction in the care provided by cuckolded males. Therefore, no evidence was found to support parental care hypothesis as a driver of EP mating in the noisy miner. Despite this, we cannot rule out that EP mating behavior may have other functions, such as increasing offspring fitness via increased heterozygosity or “genetic quality” (Griffith et al. 2002), or providing fertilization insurance in case the breeding male is infertile. SUPPLEMENTARY MATERIAL Supplementary data are available at Behavioral Ecology online. FUNDING The School of Environmental and Rural Sciences, UNE, provided financial support for this project. The project was also partly supported by the ANZ Equity Trustee Holsworth Wildlife Endowment grant. We are grateful to Farzaneh Etezadifar for her assistance during the fieldwork and Hugh Ford for suggestions on the initial draft of the manuscript. 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