Multiple paternity
Multipaternity was confirmed from genotype analysis in 5 of the 70 clutches in which we identified more than one genotyped hatchling (7%). Two fathers per female were responsible for all of the offspring sampled in each clutch. The occurrence of multipaternity was not correlated with the number of genotyped hatchlings per clutch, as multipaternity was found in clutches with counts of 2 to 20 genotyped hatchlings (Supplementary Data 7). One male was dominant in each clutch and sired between 60% and 66% of the genotyped hatchlings (Figure 5) (except in the clutch with only 2 genotyped hatchlings).
Out of these five clutches, two were laid by the same female, CMY0188 (1415_ONE8 and 1415_ONE9) (Figure 5). She laid these two clutches within 13 days in season 2014/15. They were sired by the same fathers *129 and *133, with the contribution of father *133 slightly decreasing in the second clutch (66% to 60%). In this season, eight other clutches laid by the same female were sired only by father *133, showing no multipaternity (Table 4). Three of them were laid before the clutches with multipaternity and counted 3, 17, and 6 genotyped hatchlings, respectively. The remaining 5 were laid after 1415_ONE9 and counted 8, 9, 7, 1, and 6 hatchlings (Table 4). Hatchlings from father *129 thus occurred only after several egg-laying events, possibly indicating mating in the inter-nesting interval.
Two other clutches laid one year apart were also sired by the same fathers (*62, *53) but these were from two different females, CMY1384 and CMY2419 respectively (Figure 5). For the female CMY1384, we found three nests in 2017/18 counting 1, 3, and 2 genotyped hatchlings, and multipaternity was found only in the second clutch, with father *53 absent from the other clutches. For CMY2419, 4 nests were identified in season 2018/19 (2, 2, 1, and 2 genotyped hatchlings) and father *62 was found only in the second clutch, the only one to show multipaternity (Table 4). Finally, the last clutch with multipaternity, 2021_ONE17, was laid by female CMY3468 in season 2020/21 and sired by fathers *97 and *337. Another clutch was laid by the same female after 36 days, from which two hatchlings were sampled. Only father *97 was found responsible for the genotype of these hatchlings (Table 4). Thus, multipaternity was never found in all the clutches laid by a female within a season.
We then investigated the effect of multipaternity on fitness. When comparing the fitness parameters in clutches with and without multipaternity, no correlation was found between the number of fathers and any of the three parameters (t.test p_values: 0.91, 0.95, and 0.83 for the clutch size, the total number of hatchlings, and the number of dead embryos respectively. Figures in supplementary data 4).