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Supplemental information accompanying the Behavioral Ecology and Sociobiology paper:
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Supplementary methods
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Reproductive competition and inbreeding avoidance in a primate species with habitual
female dispersal
by Linda Vigilant1, Justin Roy, Brenda J. Bradley, Colin J. Stoneking, Martha M. Robbins, Tara
S. Stoinski
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Department of Primatology, Max Planck Institute for Anthropology, Deutscher Platz 6, 04103
Leipzig, Germany vigilant@eva.mpg.de
Genetic analysis
Most samples were collected using a two-step method of short-term storage in alcohol
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followed by desiccation, but some earlier samples were simply desiccated using silica gel or
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stored in RNAlater (storage details as described in (Nsubuga et al. 2004). We extracted DNA
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using the QIAmp Stool kit (Qiagen).
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For genotyping we used 13 of the 15 loci employed previously in a study of this
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population (Bradley et al. 2005) and replaced the previously used D7s794 and D18s851 with
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D1s2130, D5s1457, and D8s1106. We used a multiplexing protocol in which all primers were
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included in an initial PCR followed by re-amplification at each single locus. Primer sequences
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and PCR reaction conditions are as previously detailed for mountain gorillas (Arandjelovic et al.
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2009). Dilutions of PCR products were combined in sets of three or four with ROX labeled
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HD400 as an internal size standard and electrophoresed on an ABI Prism 3100 Genetic Analyzer
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and analyzed using GeneMapper software v. 3.7 (Applied Biosystems).
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Whenever possible we extracted DNA and produced genotypes from more than one
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sample per individual in order to confirm attribution of the samples to particular individuals. We
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performed extensive replication of PCRs according to the amount of DNA present in the extracts
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as described in: (Arandjelovic et al. 2009) in order to guard against genotyping error, particularly
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allelic dropout and false inference of a homozygous genotype. In practice, homozygous
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genotypes were observed at least 3 times and often 6 or more times. In addition, we checked that
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genotypes of known relatives (ie., mother-offspring) were genetically compatible, and confirmed
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the sex of the individual using a PCR-based assay as previously described (Arandjelovic et al.
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2009).
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General Linear Mixed Models
We used a binomial variance model and logit link function. The log odds ratio of the
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inverse number of males was included as an offset term. The male ID, mother ID and offspring
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ID were included as multilevel random effects. All predictors were transformed to have zero
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mean and unit standard deviation. Predictors that represented timespans (age, tenure length) were
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additionally square-root-transformed prior to this step. We checked for multicolinearity among
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the predictors by computing the model matrix condition number; condition numbers greater than
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30 were taken as evidence of potentially interfering multicolinearity (Belsey et al. 1980). The p
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values were based on a likelihood ratio test, which avoids problems with p values based on a
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normal approximation (Hauck and Donner 1977). The p values were determined from an initial
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model fit using all relevant predictors, and adjusted for multiple comparisons using the Holm
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procedure (Holm 1979). After fitting the initial full model, we removed predictors stepwise until
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the Akaike information criterion was minimal (Sakamoto et al. 1986). We do not report p values
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for the predictors in these reduced models, because they may be influenced by the model
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reduction. In order to evaluate how well our best-fitting models actually explained the data, we
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plotted receiver operating characteristic (ROC) curves, using the ROCR package in R (Sing et al.
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2005). We used the area under the ROC curve as a convenient summary value (Bradley 1997).
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Supplementary results
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Genotyping and parentage assignment. The genotypes of 149 gorillas at 16 autosomal
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microsatellite loci were 95% complete. There was an average of 4.63 alleles and an expected
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heterozygosity of 0.55. The ability to determine paternity was high, with a combined non-
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exclusion probability of 0.0017 given the genotype of the mother. One locus (D4s1627) was
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found to not be in Hardy-Weinberg equilibrium after correction for multiple testing, but since the
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data consists of a structured population containing many relatives this result is not unusual and is
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not expected to affect the paternity determinations. The paternity assigments produced using
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CERVUS had high (90%) likelihood and in all cases the assigned father had no genotypic
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‘mismatches’ to the offspring and other candidate males were excluded by one or more
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mismatches.
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Of the 47 paternities reported previously (Bradley et al. 2005), we reassigned MNZ,
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formerly attributed to ZIZ, to PAB. The prior assignment was based on less complete data and
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we now find that PAB is compatible at 16 loci with MNZ and the mother and hence was
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assigned as the father by CERVUS with high likelihood while ZIZ mismatches at one of 16 loci.
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All other previous assignments were confirmed, and we amend the previous unspecific paternity
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assignment of TEG from ‘NotShinda’ to NTA. Three offspring (RIB, RAM, RUS) were
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conceived in groups containing a single silverback male and in each case he was the sire. One
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additional offspring (KEZ) was born in a group containing one silverback and one juvenile male,
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and the silverback was the sire. The other 93 offspring assigned were conceived in groups
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containing multiple silverback and potentially reproductive ‘blackback’ males. (Supplementary
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Information Table S1).
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With regard to the shares of paternity accruing to the dominants (Table 1 main text), for
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one male (BEE) we assess only three paternities from the final four years of his tenure, and
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similarly analyze paternities produced in the final seven years of the approximately eight year
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tenure of ZIZ. For the other males, we analyzed offspring produced during the entire 13 year
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tenures of SHI and TIT, respectively and during 11 years of the ongoing tenure of CAN.
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Cases of female dispersal. In the BEE group, two nulliparous females (UMC, PAS) transferred
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to different groups in 1999 at the ages of 9.4 and 8.5. IZZ did not produce offspring in her natal
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group (BEE) but only after dispersing at the relatively late age of 12.8. In Group 5, the daughters
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of the dominant ZIZ did not reach typical dispersal age before the death of the dominant and
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subsequent formation of PAB and SHI groups. Two females (UKU, MHW) were fathered by
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PAB in 1993 during a period for which it is unknown whether PAB or CAN was the dominant
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male, and both dispersed from their natal group, which by then featured CAN as dominant male,
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by the ages of 7.7 and 7.3. Both daughters of SHI (KAN, KRD) that were of dispersal age
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stayed and conceived in the natal group.
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Supplementary References
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Arandjelovic M, Guschanski K, Schubert G, Harris TR, Thalmann O, Siedel H, Vigilant L (2009) Two-
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step multiplex polymerase chain reaction improves the speed and accuracy of genotyping using
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DNA from noninvasive and museum samples. Mol Ecol Resour 9:28-36
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Belsey DA, Kuh E, Welsch RE (1980) Regression Diagnostics: Identifying Influential Data and Sources
of Collinearity. John Wiley & Sons, Hoboken
Bradley AP (1997) The use of the area under the roc curve in the evaluation of machine learning
algorithms. Pattern Recogn 30:1145-1159
Bradley BJ, Robbins MM, Williamson EA, Steklis HD, Steklis NG, Eckhardt N, Boesch C, Vigilant L
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(2005) Mountain gorilla tug-of-war: Silverbacks have limited control over reproduction in
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multimale groups. Proc Nat Acad Sci USA 102:9418-9423
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Hauck WW, Donner A (1977) Walds test as applied to hypotheses in logit analysis. J Am Stat Assoc
72:851-853
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Holm S (1979) A simple sequentially rejective multiple test procedure. Scand J Stat 6:65-70
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Nsubuga AM, Robbins MM, Roeder AD, Morin PA, Boesch C, Vigilant L (2004) Factors affecting the
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amount of genomic DNA extracted from ape faeces and the identification of an improved sample
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Sakamoto Y, Ishiguro M, Kitagawa G (1986) Akaike information criterion statistics. D. Reidel, Dordrecht
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Sing T, Sander O, Beerenwinkel N, Lengauer T (2005) ROCR: visualizing classifier performance in R.
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Bioinf 21:3940-3941.