Introduction

In most species with parental care, the investment of the two parents is unequal, with the mother usually contributing more than the father to parental duties. Differences in parental investment are rooted in the initial investment in gametes, with females producing larger eggs than the males' sperm cells (i.e. anisogamy). This asymmetric situation is the source of conflict between the sexes over their investment [1]–[3]. The most extreme form of conflict is when one parent decides to abandon its progeny and its partner. The decision to care for or desert offspring in search of new mating opportunities has long been recognized as a source of conflict between genders. Whether a parent should stay or leave depends on the probability of finding a new mate and whether the presence of both parents is required to successfully raise progeny [4], [5]. When the presence e.g. of the father is not necessary to ensure offspring survival, males desert soon after copulation, because caring for offspring entails the cost of lost mating opportunities [6]. Because males can more quickly replenish their relatively cheaper sperm than females can replace their costly eggs, males are usually predicted to invest more heavily in traits that increase mating success rather than investment in offspring care. However, if adult sex ratio is evenly balanced, males who are ready to mate may be unable to find a currently fertile female partner, depending on the degree of breeding seasonality of the species. Indeed, on average, males will have to wait for females to become sexually available. Hence, it may be advantageous for them, in term of fitness prospect, to take care of their offspring (instead of deserting), during periods when females are not available for mating.

Although in most of the species with parental care, the female is the primary caregiver, there are notable exceptions. For example, in birds, many species are monogamous with males participating in parental care (about 90% of bird species, [7]). With the exception of a few polygamous species where males do not participate in parental care but compete to secure more than one female, monogamous males can trade-off their investment in offspring rearing against the search of extra-pair copulations by decreasing their level of care [8]. The situation can be even more complex in species that produce several broods per year. Indeed, the temptation is high to abandon the brood to its mate in order to start a second breeding attempt with a mate free of parental duties. This situation is particularly interesting because it is not evident at first sight which of male or female should desert its brood. Depending on the costs and benefits of desertion, one of the two sexes may be more likely to desert. This decision will depend on sex ratio of available mates and on which parent is most useful to pursue offspring rearing, as well as resources availability (e.g. [9], [10]). If more males than females are not breeding and hence available to produce a brood late in the season, females may be more tempted to desert their brood and start a new one [11], as has been shown for example in the rock sparrow (Petronia petronia) [12]. Furthermore, if the fitness of offspring is more sensitive to male than female care, males may be less tempted to desert than females. This is the case in the barn owl (Tyto alba) and Tengmalm's owl (Aegolius funereus) in which the presence of the mother is not mandatory to raise the offspring between the period when they can consume food without maternal help and independence [13]–[15]. Females frequently desert their mate and offspring halfway through the rearing period to produce a 2^nd annual brood with another male. The first male may thus frequently copulate during offspring rearing in case his female deserts to produce a 2^nd annual brood with another male. Although copulation frequency is maximal during egg laying (on average more than one copulation per hour), barn owls can continue to copulate during the entire rearing period (but at a lower rate) particularly when owls are planning to produce a 2^nd annual brood [16], [17]. This behaviour may increase the likelihood that the male sires some of the offspring of the female's 2^nd brood in case she suddenly deserts him [18]. Although in raptors and owls extra-pair paternity is usually very low (less than 1% of the broods contain extra-pair young, i.e. young sired by another male than the one that feeds them [19]), we predict that 2^nd annual broods of deserting females entail a number of young sired by the male with which these females produced the 1^st annual brood. We tested this prediction with paternity analyses of the 1^st and 2^nd annual broods of female barn owls that produced these two successive broods with a single or with two different males.

Materials and Methods

Linkage between markers

We tested for genotypic disequilibrium between loci and deviation from Hardy–Weinberg equilibrium was tested to check for the presence of allelic dropouts, null alleles, substructure and inbreeding. Deviations from random mating within populations (F_IS) per locus and sample were computed with a bootstrap procedure of 120 randomizations. F_IS values were not significantly different from zero (Table 1) and no linkage disequilibrium between pairs of loci was detected (all P-values < adjusted 5% level). All summary statistics and tests were computed using FSTAT Version 2.9.3 [23]. Finally, significance values were corrected for multiple tests using the sequential Bonferroni method [24].

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Table 1. Summary statistics in the adult barn owl population for all six microsatellite loci.

https://doi.org/10.1371/journal.pone.0080112.t001

Results

All of the 455 assigned offspring had a level of confidence for parentage pair assignment higher than 95% and zero mismatches with their assigned biological father. However, concerning three nestlings of 1^st annual broods, the discrimination power between the two potential fathers was very low, due to similarities in their genotypes (i.e. the nestlings had zero mismatches with both of their potential fathers). We therefore excluded them from the results.

We detected extra-pair nestlings in two 2^nd broods produced by deserting females who produced their 2^nd annual brood with another male than the one with whom they produced the 1^st annual brood. Paternity analyses showed that the males at the 1^st breeding attempt sired the six extra-pair young (3 out of 8 young in one brood and 3 out of 7 young in the other brood). The two nests of these deserting females were located at 980 m and 1500 m distance, respectively, and at the first nest the offspring were aged 44 and 40 days at the time when their mother laid her first egg at the 2^nd annual clutch with a new mate. In comparison, the mean distance between the two nests of deserting females, for which all the offspring had been sired by the social male, was 3.3 (±0.5 SE) km. All 219 offspring from 49 1^st annual broods were sired by the social father as well as all 93 offspring from 20 2^nd annual broods when the mother bred with the same male as at the 1^st annual brood.

Discussion

Extra-pair paternity is very low in the barn owl as shown in the present study (six out of 455 nestlings) and another study (one out of 211 nestlings) using an independent sample of broods [19]. Interestingly, six of these seven extra-pair young were found in two 2^nd annual broods of females that deserted their first mate (present study) and one young was raised in the 1^st annual breeding attempt of the female [19]. This is consistent with the hypothesis that copulations performed while rearing the offspring can allow males to increase their paternity in case their female deserts to start a 2^nd annual clutch with another male [18], [27]. From a male point of view, selection may have promoted the evolution of a high copulation frequency to increase the fitness of males who continue to take care of the offspring at the 1^st annual breeding attempt while their female produce a 2^nd annual brood with another male. From a female point of view, copulating during offspring rearing may be a strategy to convince her male that she will produce the 2^nd annual brood with him. This behaviour could induce him to forage intensely not only for the brood but also for his female, who needs extra energy to produce eggs of the 2^nd clutch [17]. Another scenario posits that high quality females can afford to desert the 1^st brood and are also better to secure extra-pair copulations. This is consistent with the observation that the distance between the two nests of deserting females, for which some offspring at the second nest were sired by the 1^st male, was relatively short (less than 1500 m while the distance between nests without multiple paternity was on average 3.3 km). Therefore, multiple paternity may occur only if the distance between the two females' nests is not too large, potentially indicating that females do not store sperm from the 1^st male to sire eggs at the second nest. If so, females would have to return to the 1^st nest to copulate with the 1^st male or, alternatively, the 1^st male would have to visit her female at her 2^nd nest. An anecdotal personal observation showed that deserting females can indeed continue to visit her 1^st nest.

In the barn owl, females that intend to produce a 2^nd annual brood benefit from deserting their first brood and breed again with a new mate, since deserting females produce their 2^nd annual brood two weeks earlier than non-deserting females [13]. However, the new mates of deserting females are usually yearlings, and hence probably males of low quality, which could explain why deserting females produced a similar number of fledglings as non-deserting females even though they laid significantly more eggs implying that nestling mortality is higher at the 2^nd brood of deserting than non-deserting females [13]. As a consequence, deserting females may derive genetic benefits if in the 2^nd annual brood some offspring have been sired by the male with whom they produced the 1^st annual brood.

The level of multiple paternity that we observed in the barn owl is comparable to the levels observed in other polyandrous bird species where females produce successive clutches with several males who incubate them. In the comb-crested jacana (Irediparra gallinacea) only 2.8% of the nestlings were sired by another male than the one who incubated the eggs [28], in the Eurasian dotterel (Charadrius morinellus) only 4.6% [29] and in the the red phalarope (Phalaropus fulicarius) only 6.5% [30]. This rate is much lower than in some lek-mating species (45% in the wild turkey [Meleagris gallopavo] [31]), or in monogamous species (11.1% on average [32]). Because in polyandrous species males invest so much effort in reproductive activities, selection is intense to avoid being cuckolded. This can select for a high copulation frequency (as observed in the barn owl [17], [33], [34]). However, because the second male of deserting females could not prevent their female to copulate with her first male, other behaviour may have evolved to reduce the risk of cuckoldry. For instance, in the wattled jacana (Jacana jacana) males remove the first egg of their clutch except at the first annual breeding attempt [35]. This suggests that males from polyandrous species could constrain female extra-pair mating behaviour by imposing high fertility costs to females. If this behaviour also occurs in the barn owl it could explain why we found extra-pair young in only two out of 29 second broods of deserting females. Another possibility to explain the relatively low level of multiple paternity is that the distance between the two successive nests of deserting females was relatively high preventing females to visit their first male.

Acknowledgments

We thank Andrea Pilastro and an anonymous referee for useful comments.