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
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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)