1 SUPPLEMENTARY MATERIALS AND METHODS (a) Parentage analysis and assignment assumptions Although more than 97% of the birds in the study population were sampled during the study, there was an average of 0.47 unmarked males and 0.35 unmarked females per group per year that were never captured. 36 of 40 (90%) social mothers identified from behavioral observations at the 100 nests from which offspring were sampled were captured. These females accounted for 233 of 247 (94%) offspring in the genetic dataset. Parentage analysis revealed that the social mother was the genetic mother for all 233 of 233 (100%) offspring. Because there was no evidence of intraspecific brood parasitism, it was assumed that the genetic mother of the remaining 14 offspring was the unsampled social mother. In the parentage analysis, a known parent (mother) was included for all but 14 of 247 (6%) offspring; the 14 samples represent those cases where the social mother of a nest was not captured. All of the males in the entire study population that were alive in a given year were included as candidate fathers in the analysis. CERVUS unambiguously identified a single genetic father for 195 of 247 (79%) offspring with zero allelic mismatches at an 80% or 95% confidence level; 11 of 195 (6%) fathers were assigned at an 80% confidence level and 184 of 195 (94%) fathers were assigned at a 95% confidence level. The remaining 52 of 247 (21%) offspring could not be unambiguously assigned a father from the initial CERVUS analysis and fell into one of three categories: (i) cases where there were two potential candidate fathers; (ii) cases where there were no potential candidate fathers, but where the social father was not captured; and (iii) cases where there were no potential candidate fathers, but where the social father was captured. First, there were 20 of 247 (8%) offspring for which CERVUS identified two potential candidate fathers with similar LOD scores and zero allelic mismatches at all 15 loci 2 (category i). In all but one case, one of the candidate fathers was the social father. To assign a single candidate father to each of these 20 offspring, three methods were used: (i) the two loci that showed evidence of null alleles (Table S1) were used to help visually exclude one of the candidates; (ii) birds less than 2 yrs of age were assumed to have not bred because there was only one known case of a bird less than 2 yrs old siring any offspring in the 195 cases where paternity was unambiguously assigned; and (iii) birds were assumed to have avoided inbreeding with their mother because there were no cases of inbreeding in the 195 cases where paternity was unambiguously assigned (there were only two cases of potential inbreeding where the second candidate was related to the first and greater than 2 yrs old). Using these methods, paternity was assigned to the male who sired the other nestlings within the brood in 18 of 20 (90%) cases, and to an extrapair male in the remaining two cases. Second, there were 18 of 247 (7%) offspring for which CERVUS could not assign a candidate father, but whose social father was not captured (category ii); only 4 of 38 (11%) males identified from behavioral observations as the social father were not captured. In each of these cases, the unsampled social father was assumed to be the genetic father. Third, there were 14 of 247 (6%) offspring for which CERVUS could not assign a candidate father, but whose social father was captured (category iii). Although there was an average of 0.47 unmarked males per group per year, the genetic father was assumed to be a male from outside the group for these remaining 14 offspring. Thus, a genetic father was assigned to 233 of 247 (94%) offspring, and the genetic fathers of the remaining 14 of 247 (6%) offspring were scored as extra-group males. Because in the parentage analysis I assumed that offspring who could not be assigned a father were sired by extra-group extrapair males (category iii), even though some groups contained unsampled males in some years, I used a reduced dataset of offspring born into groups 3 in which all of the males were sampled in that year to test this assumption. Specifically, I examined genetic heterozygosity in social parents at nests with extra-group extrapair young and found that the results from the reduced dataset were similar to those of the full data set; standardized heterozygosity (GLMM: F1,11 = 2.33, P = 0.16) and internal relatedness (GLMM: F1,11 = 1.96, P = 0.19) did not differ between the social parents. 4 SUPPLEMENTARY RESULTS AND DISCUSSION (a) Copulations Only three copulations were observed in more than 10,000 hr of observation during the five year study. One copulation occurred a few weeks before the breeding season, one occurred at the beginning of the breeding season, and one occurred a few weeks into the breeding season. All three copulations occurred between 10.00 and 14.00 hrs and took place on the ground. One copulation was between a pair-bonded male and female, but the other two were extrapair copulations. In the only case where either the male or female were observed prior to the copulation, a female solicited an extrapair copulation from an unrelated individual who had been helping to feed nestlings at her nest by spreading her wings slightly, angling her body towards the ground at a 45º angle, and quivering her tail and wings while rapidly dilating her eyes. Similar solicitation behavior has been reported in the closely related chestnut-bellied starling, Lamprotornis pulcher (Fry et al. 2000). The other extrapair copulation occurred between an unmarked female—presumably from another social group and not a floater, since it was during the breeding season after all dispersing females had joined groups—and a marked male on his group’s territory. Although these limited observations suggest that females are capable of soliciting copulations and seeking extrapair fertilizations, as has been observed in other species of starlings (Eens & Pinxten 1990; Fry et al. 2000), they do not establish whether patterns of extrapair paternity are the result of male- or female-initiated reproductive strategies (Westneat & Stewart 2003). 5 SUPPLEMENTARY TABLES Table S1. Predictions about female extrapair mate choice for the direct benefits, indirect, genetic benefits, and fertility insurance hypotheses (sensu Griffith et al. 2002). Hypothesis Direct benefits Indirect, genetic benefits Predictions Where extrapair mates are from Within group Within and outside group When to copulate with extrapair mates1 When fewer offspring helpers available What effect on offspring Extrapair offspring genetically similar to within-pair offspring Good genes2: Higher quality males Good genes2: When paired to poor quality males Good genes2: Extrapair offspring more fit than within-pair offspring Genetic diversity: Random Genetic diversity: Random Genetic diversity: Extrapair offspring genetically similar to within-pair offspring Genetic compatibility: More genetically compatible males Genetic compatibility: When paired with genetically similar males or When paired to males with relatively lower heterozygosity than themselves Genetic compatibility: Extrapair offspring more heterozygous than within-pair offspring More fertile males3 When paired to an unfertile male Extrapair offspring genetically similar to within-pair offspring 1 Although year-to-year variation in food availability, as well as the level of paternal care by the pair-bonded male, could directly influence a female’s threshold for seeking extrapair fertilizations (Shellman-Reeve & Reeve 2000), year-to-year variation in rainfall did not influence extrapair paternity rates in superb starlings (Rubenstein In revision), and the level of paternal care did not vary among years or between nests with and without extrapair offspring (Rubenstein 2006). 2 The good genes hypothesis was not tested in this study because (i) not all extra-group extrapair males were captured and (ii) offspring quality was not measured, since more than half of the nests containing extrapair offspring were depredated before fledging. 3 The fertility of extrapair males was not measured. Fertility insurance Within and outside group Which extrapair mates to copulate with Non-breeders who could become helpers 6 Table S2. Summary of allele frequencies for a panel of 17 microsatellite markers developed for the superb starling, Lamprotornis superbus. Locus No. of alleles Product size range (bps) HO HE Excl (1) Excl (2) SS1-6 9 195 – 213 0.83 0.81 0.47 0.64 SS2-10* 22 313 – 513 0.38 0.89 0.64 0.78 SS1-11* 7 180 – 191 0.39 0.80 0.43 0.61 SS1-12 6 207 – 217 0.21 0.21 0.02 0.11 SS1-15 4 307 – 316 0.49 0.47 0.11 0.27 SS2-32 5 246 – 254 0.40 0.38 0.08 0.21 SS3-42C 22 138 – 188 0.89 0.88 0.61 0.76 SS2-46 4 183 – 189 0.24 0.26 0.03 0.14 SS2-49 4 114 – 120 0.48 0.54 0.15 0.27 SS2-68 6 142 – 151 0.55 0.55 0.15 0.27 SS2-71B 7 329 – 341 0.74 0.70 0.28 0.45 SS2-76 7 154 – 171 0.15 0.14 0.01 0.08 SS2-83 11 144 – 180 0.80 0.75 0.38 0.56 SS2-106 5 284 – 300 0.35 0.35 0.06 0.19 SS2-121 5 167 – 173 0.73 0.75 0.34 0.51 SS2-130 5 260 – 269 0.53 0.56 0.16 0.27 SS2-132 8 224 – 252 0.38 0.38 0.08 0.22 Combined 15 loci 7.20 0.52 0.52 0.975 0.999 The number of alleles and the product size range and for each locus are given. Observed (HO) and expected (HE) heterozygosities, as well as the exclusion probabilities for the first parent (Excl [1]) and the second parent (Excl [2]) were calculated using the program CERVUS. Null alleles were detected for two loci (indicated by an asterisk) after more than 350 individuals were genotyped, so these markers were excluded from the primary parentage analysis. Analyses with the remaining 15 loci were conducted on 447 individuals and 100% of alleles were scored. 7 Table S3. Results from simulations to determine parentage assignment success rates when one or neither parents were known. Confidence level Neither parent known One parent known Success rate Success rate Success rate Success rate (without R) (R = 0.25) (without R) (R = 0.25) 80% confidence 100% 100% 100% 100% 95% confidence 76% 57% 99% 93% To determine parentage assignment success rats at 80% and 90% confidence levels when one ore neither parents were known, two simulations were run using CERVUS, each for 100,000 cycles. The first simulation assumed no relatedness between candidate parents, whereas the second assumed a relatedness of 0.25 between offspring and candidate parents and estimated five potential relatives in the pool of candidate parents. Both simulations assumed a 1% error rate in typing, the default value for CERVUS, as well as 20 candidate parents. A total of 100% of loci were genotyped, and based upon field data, an estimate of 95% of the candidate parents sampled was used in the simulations. 8 SUPPLEMENTARY REFERENCES Eens, M., & Pinxten, R. 1990 Extra-pair courtship in the starling, Sturnus vulgaris. Ibis 132, 618-619. Fry, C. H., Keith, S., & Urban, E. K. 2000 The Birds of Africa. San Diego: Academic Press. Griffith, S. C., Owens, I. P. F., & Thuman, K. A. 2002 Extra pair paternity in birds: a review of interspecific variation and adaptive function. Mol. Ecol. 11, 2195-2212. (doi:10.1046/j.1365-294X.2002.01613.x) Rubenstein, D. R. 2006 The evolution of the social and mating systems of the plural cooperatively breeding superb starling, Lamprotornis superbus. Ph.D. Dissertation, Ithaca, NY: Cornell University. Rubenstein, D. R. In revision Territory quality drives intraspecific patterns of extrapair paternity. Behav. Ecol. Shellman-Reeve, J. S., & Reeve, H. K. 2000 Extra-pair paternity as the result of reproductive transactions between paired mates. Proc. Roy. Soc. Lond. B 267, 2543-2546. (doi:10.1098/rspb.2000.1318) Westneat, D. F., & Stewart, I. R. K. 2003 Extra-pair paternity in birds: causes, correlates, and conflict. Ann. Rev. Ecol. System. 34, 365-396. (doi:10.1146/annurev.ecolsys.34.011802.132439)