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Providing parental care entails variable mating opportunity costs for male Temminck’s stints
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Behavioral Ecology and Sociobiology
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Robert L. Thomson, Veli-Matti Pakanen, Diane M. Tracy, Laura Kvist, David B. Lank, Antti Rönkä and
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Kari Koivula
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Corresponding Author: Robert L. Thomson (robtho@utu.fi). Current address: Section of Ecology,
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Department of Biology, University of Turku, FI-20014 Turku, Finland
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Appendix B: Parentage analyses: a step by step description
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Parentage analyses were done separately for each main breeding site assuming no movement between sites.
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When simulating critical values for LOD values for 80% and 95% confidence levels, we assumed 1%
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typing error rate. Depending on the breeding site 70 to 90 % of candidate males and 35 to 70% of candidate
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females were assumed to have been sampled.
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Due to the Temminck’s stint breeding system, no observational data exists to identify the non-incubating
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parent. Consequently, analyses started by assigning incubating parents to each nest. Nests were then split
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by incubator sex and analyzed separately. Offspring were sorted by sex because hemizygous loci can only
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be used for male offspring, which receive alleles from both parents. Analysis of female offspring was done
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with six loci (Calp2, Ruff6, 49F6, Pgt83, Hru2 and Gme3).
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Analyses were done as either maternity or paternity tests with the incubating sex known. Potential parents
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included all sampled adults in a particular breeding site, including all individuals born/present in earlier
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years, despite not being confirmed at the site in that year. All mismatches with known parents against their
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chicks were checked from the ABI electropherograms to rule out possible scoring errors.
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When analyzing male offspring, we had to consider that allele frequencies were biased by the fact that
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female genotypes were included as homozygous despite having only one allele for hemizygous loci. This
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may affect estimates of LOD scores for parents because CERVUS does not consider sex-linked loci in the
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calculation of allele frequencies. Paternity of incubating parents was thus judged on the basis of parent-
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offspring mismatches. Mixed paternity was confirmed in cases of multiple mismatches. One mismatch was
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ruled a mistyping error (11 cases) if there was no data to suggest otherwise, for example a neighboring
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male with zero mismatches (observed in one brood) or when a male siring other chicks in that brood
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perfectly matched a chick (observed in one brood). Incubating males had on average 2.5 mismatches (range
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1 – 4) with EP chicks.
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For offspring with incubating parents that did not match (EPs) and offspring from nests with unidentified
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incubating parent (mostly female incubated nests, eight sampled offspring), the parent pair-test with the
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known sexes approach was used. Here all possible combinations were tested, including individuals from all
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breeding sites.
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When assessing paternity in female incubated nests and EP chicks in male incubated nests, we examined
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also the trio-mismatches (i.e. combinations of father-mother-offspring alleles) and associated LOD scores.
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Because the number of microsatellites used gave low resolution in finding parents for female offspring,
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those not assigned to any male were reanalyzed with data including hemizygous loci. We manually checked
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each male genotype (allele sizes) of all potential parents against female offspring for mismatches in the sex-
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linked loci. By doing so, we confirmed paternity in 27 cases where there were zero mismatches for two
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potential fathers. Crosschecking paternity within broods resulted to the same conclusions.
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Nine chicks had two potential fathers with zero mismatches in all loci. In these cases, we assigned paternity
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based on LOD scores and information on paternity of other chicks in that brood. In most cases (7), only one
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male fit perfectly to all other offspring. Following parsimony, that male was also considered to be the father
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of the offspring in question. The last two were assigned on the basis of territory location and LOD scores.