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The adaptive function of female extrapair mating in socially monogamous passerines is currently debated. In the bluethroat (Luscinia s. svecica), a previous study showed that offspring sired by extrapair males had a higher cell-mediated immunity than their within-pair half siblings, suggesting an immunogenetic benefit of extrapair mating in this species. Here, we expanded that dataset with two more years and investigated the association between extrapair paternity and microsatellite multilocus heterozygosity, in addition to cell-mediated immunity. We found that extrapair offspring were more heterozygous than their within-pair half siblings, and corroborated the previous finding of enhanced cellular immunity in extrapair offspring in this four-year dataset. The increased heterozygosity among extrapair offspring appeared to be a result of extrapair mates being less genetically similar than pair mates, and also less genetically similar than expected by random choice. Together with previous findings in this species, showing that the majority of females participate in extrapair copulations, our results indicate a postcopulatory cryptic female choice of genetically dissimilar males. The enhanced cellular immunity and increased heterozygosity were not related to each other, and hence our results indicate two independent genetic benefits of extrapair mating in the bluethroat.
In some species, females may gain nongenetic (direct) benefits by being allowed to forage inside the extrapair male's territory, by being helped by the extrapair male in predator defense (Gray 1997) or brood care (Blomqvist et al. 2005), or by insuring successful fertilization of her eggs (Sheldon 1994). Alternatively, females may gain genetic (indirect) benefits, by producing extrapair offspring of higher quality than within-pair offspring (e.g., Johnsen et al. 2000; Foerster et al. 2003; Garvin et al. 2006; Stapleton et al. 2007). In socially monogamous species, female mate choice can be constrained by the number of single males, and extrapair copulations may be a strategy to modify their initial choice (Møller 1992). For example, the occurrence of extrapair offspring is associated with high genetic similarity between social pairs in several passerine species (Freeman-Gallant et al. 2003; Eimes et al. 2005; Tarvin et al. 2005; Freeman-Gallant et al. 2006), and hence females seem to avoid the negative effects of inbreeding by engaging in extrapair copulations (Pusey and Wolf 1996). Alternatively, rather than only avoiding inbreeding, the females may be targeting specific extrapair males to maximize the genetic quality of their offspring, either by choosing high-quality extrapair males (the good genes hypothesis), or by choosing extrapair males whose genome best complements their own (the compatible genes hypothesis).
A powerful way to test for genetic benefits of extrapair fertilizations is to compare within-pair and extrapair offspring raised in the same nest, that is, maternal half siblings. These nestlings are exposed to the exact same environmental conditions, and originate from the same maternal genotype. Thus, any difference between them should be caused by the differential paternal genetic contribution (Sheldon et al. 1997). In addition, comparing paternal half siblings can reveal if a genetic benefit is due to good genes per se or compatible genes (e.g., Johnsen et al. 2000; Garvin et al. 2006): whereas a genetic effect through compatible genes also predicts a difference between paternal half siblings, an effect of good genes does not.
Individual heterozygosity is a prime example of a compatibility benefit of extrapair mating, because the level of heterozygosity to a large extent depends on the relative genetic similarity of an individual's parents (Brown 1997; Charlesworth and Charlesworth 1999). Heterozygosity is positively related to survival and reproductive success in many taxa (Coulson et al. 1998; Coltman et al. 1999; Amos et al. 2001; Hansson et al. 2001; Tregenza and Wedell 2002; Charpentier et al. 2005; Pujolar et al. 2005). In passerines, there are several examples that heterozygosity correlates positively with survival, territory size, song diversity, male plumage characters, clutch size, hatching success, fledging success, and fertilization success (Hansson et al. 2001; Foerster et al. 2003; Cordero et al. 2004; Seddon et al. 2004). Furthermore, extrapair offspring have been found to have a higher heterozygosity than their maternal half siblings in blue tits Cyanistes caeruleus (Foerster et al. 2003) and tree swallows Tachycineta bicolor (Stapleton et al. 2007).
In the socially monogamous bluethroat Luscinia s. svecica, a previous study revealed that extrapair offspring have an increased cell-mediated immunity, as expressed by the swelling response to phytohemagglutinin (PHA), compared to both their maternal and paternal within-pair half siblings, lending support to the compatible genes hypothesis (Johnsen et al. 2000). In the present study, we expanded that dataset with two more years, and investigated the effect of extrapair paternity on heterozygosity, in addition to cell-mediated immunity. We employed microsatellite markers to calculate multilocus heterozygosity and pairwise genetic similarity, and examined the association between these variables and extrapair paternity in the four-year dataset on bluethroat nestlings.
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Extrapair offspring had a significantly higher heterozygosity than their maternal within-pair half siblings. The increased heterozygosity appeared to be a result of extrapair mates being less genetically similar than within-pair mates, suggesting a selection for genetically dissimilar partners in the bluethroat. The previously found enhanced cell-mediated immunity in extrapair offspring was confirmed in this expanded dataset, and moreover, it seemed to be independent of the effect of increased heterozygosity. Thus, extrapair mating seems to entail multiple genetic benefits in the bluethroat.
The results of our half-sibling comparisons corroborate the conclusion of Johnsen et al. (2000) that females benefit by mating with a genetically compatible extrapair male rather than one with good genes in an absolute sense. First, the enhanced immune response of extrapair offspring was evident in both maternal and paternal half-sibling analyses, showing that it is the combination of male and female genotypes that produce the enhanced response rather than the male genotype alone. Second, heterozygosity depends more strongly on the relative genetic similarity of an individual's parents than on paternal heterozygosity per se (Table 4; Brown 1997; Charlesworth and Charlesworth 1999), and accordingly we found that the females were less genetically similar to the extrapair sires than their pair males in our dataset.
Extrapair offspring showed both an enhanced immunocompetence and an increased heterozygosity compared to their maternal half siblings (Fig. 1A). This could theoretically be due to a causal relationship between heterozygosity and PHA response. However, a correlation between the difference in PHA response and the difference in heterozygosity between half siblings did not reveal a significant relationship. In some broods, the extrapair offspring were more immunocompetent, in others they were more heterozygous, and in some broods they were both more immunocompetent and more heterozygous (Fig. 2A). Hence, there appears to be two independent genetic benefits of extrapair mating in this species. Moreover, in only a few broods, the extrapair offspring were both less heterozygous and less immunocompetent (Fig. 2A, lower left quadrant), indicating that the females in most cases obtained at least one of the genetic benefits.
In this study, we expanded the dataset in Johnsen et al. (2000) with two more years, and corroborated the effect of paternity on offspring PHA response when considering all four years combined. However, the difference between maternal half siblings was not significant when only considering the two most recent years alone. This may be attributed to the lower sample sizes in these two years, or possibly a genotype × environmental interaction. Garvin et al. (2006) found an effect of paternity on offspring PHA response in only one of two years in the common yellowthroat (Geothlypis trichas), and discovered that the effect was only evident in the coldest of the two years. Several studies have investigated PHA response among maternal half siblings, and failed to find a significant effect of paternity (Kleven and Lifjeld 2004; Kleven et al. 2006; Edly-Wright et al. 2007). Thus, it appears that this benefit may be both context and/or species dependent.
The higher level of heterozygosity among extrapair offspring in the present study is similar to the findings of Foerster et al. (2003) and Stapleton et al. (2007), who found that extrapair offspring were more heterozygous than their maternal half siblings in the blue tit and tree swallow, respectively. However, in those studies the increased heterozygosity among extrapair offspring was caused entirely by unknown sires, that is, males not caught by them during the field season, and most likely not breeding in their study areas. In the blue tit, this seemed to result from a local genetic structure in which genetic similarity decreased with increasing distance (Foerster et al. 2003, 2006). In our study however, local extrapair males increased both the heterozygosity as well as the immunocompetence among extrapair offspring (Fig. 3). In the bluethroat, most extrapair males are close neighbors (Johnsen et al. 2001, F. Fossøy, A. Johnsen, and J.T. Lifjeld. unpublished data), and they appear to be a sufficient sample for the females to obtain a genetically compatible male.
The difference in individual heterozygosity between paternal half siblings in the bluethroat was not significant, despite the fact that the males were significantly less genetically similar to the extrapair female than to their own pair female (Fig. 1B). This may in part be explained by the relatively lower sample size of paternal compared to maternal half siblings (N= 45 vs. N= 79), in combination with the difference in calculation of heterozygosity and genetic similarity. When calculating offspring heterozygosity, we only use the information from two alleles at each locus in one individual (Coltman et al. 1999), whereas for genetic similarity we use four alleles from two individuals at each locus, and furthermore, also control for the allele frequency for each allele in the population (Queller and Goodnight 1989). However, it is noteworthy that for both the maternal and paternal comparisons of offspring heterozygosity, the direction was positive in all four years (Figs. 1A.2 and 1B.2, respectively).
Heterozygosity may be an important determinant of fitness, either because genetically depauperate individuals are more vulnerable to the negative effects of deleterious recessive alleles or because heterozygous individuals are more fit than homozygous ones due to genetic overdominance (Charlesworth and Charlesworth 1987). We found that heterozygous males showed an increased fertilization success in some years and produced heavier and more immunocompetent offspring (Table 5). The latter association appeared to result from a “good parent” effect because it was the heterozygosity of the attending male, not the genetic father, which was important for offspring immunocompetence. We also investigated the contribution of each separate microsatellite marker to the heterozygosity-fitness correlations, and found no evidence of individual markers being responsible for the relationship. Hence, it is more likely that the heterozygosity-fitness correlations are caused by a genome-wide heterozygote advantage rather than a heterozygote advantage at a specific locus (David 1998; Hansson and Westerberg 2002). In other passerines, heterozygosity is found to correlate positively with a number of fitness-related variables (Hansson et al. 2001; Foerster et al. 2003; Cordero et al. 2004; Seddon et al. 2004). Hence, a high level of heterozygosity is likely to bring an increase in fitness and extrapair offspring should therefore perform better than their maternal half siblings. Contrary to our expectations, homozygous old females laid earlier than heterozygous old females. Assuming that laying early is positive for fitness, this may suggest that homozygous females had an advantage by getting an early start of breeding. However, the fact that old heterozygous females tended to produce offspring with higher immunocompetence suggests that any advantage of laying early may be offset by a disadvantage in terms of lower offspring quality for homozygous females.
Genetically similar pairs were more likely to produce extrapair offspring than less genetically similar pairs, which resulted in within-pair offspring from nonmixed paternity broods being significantly more heterozygous than within-pair offspring from mixed paternity broods (Fig. 4B). Furthermore, there appeared to be a directional choice of genetically dissimilar extrapair partners. How then are females able to select genetically dissimilar males? Either the females have to recognize the genetic similarity to their social male and potential extrapair males in the population and then decide whether to copulate extrapair or not, or else they have to “blindly” copulate with more than one male and rely upon a postcopulatory choice of sperm (Eberhard 2000; Birkhead and Pizzari 2002; Bernasconi et al. 2004). In a precopulatory female choice scenario, only females paired with less-compatible males are expected to engage in extrapair copulations, whereas in a cryptic postcopulatory choice scenario, all females may benefit by mating with more than one male to increase the probability of being fertilized by a compatible male. In the bluethroat, recent evidence suggests that most females copulate with extrapair males although not all of them produce extrapair offspring (Fossøy et al. 2006), lending support to the postcopulatory choice mechanism rather than a precopulatory choice based on phenotypic cues. We therefore hypothesize that the females copulate readily with extrapair males, in addition to their pair male, and that the least genetically similar male has the highest probability of fertilizing the eggs (i.e., a genetically loaded raffle sensu Ball and Parker (2003)). This does not require any a priori female knowledge of the genetic similarity to either the pair male or neighboring males. Males that are genetically dissimilar to their pair female will therefore have a high chance of obtaining paternity, whereby female extrapair copulations may not result in extrapair fertilizations. In addition, there may be a stochastic effects of sperm numbers (Parker 1990), as pair males are likely to have a sperm competition advantage through higher copulatory access. This could explain why most pair males sired one or more offspring in most broods. How the actual postcopulatory mechanism works is however unknown, but an intriguing possibility is that it results from an interaction between particular substances on the surface of sperm and egg (Vacquier 1998; Palumbi 1999; Evans 2000). Alternatively, selection might take place within the female reproductive tract (Birkhead and Brillard 2007).
In summary, we found a selection for genetically dissimilar mates in the bluethroat, resulting in extrapair offspring being more heterozygous than their within-pair half siblings. Moreover, this effect appeared to be independent of the enhanced immune response among extrapair offspring in this species. Our results therefore indicate that female bluethroats obtain two different compatible genes benefits via two different genetic pathways.