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ELECTRONIC SUPPLEMENTARY MATERIAL
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Supplemental methods
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Study species. Spotted hyenas are large-bodied, long-lived carnivores that live in social groups
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called clans, which may contain over 100 members [1]. Clans are comprised of one to several
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matrilineal kin groups, each containing multiple adult females and their young [2]. In addition,
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each clan also contains one to several adult immigrant males that dispersed from other clans [2].
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Clan-mates defend a common group territory in which all members feed mainly on large and
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medium-sized ungulates they kill themselves [3]. Clans are structured by rigid linear dominance
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hierarchies in which an individual’s rank position determines its priority of access to food,
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thereby influencing virtually every aspect of its biology [1]. In this species, dominance rank
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among animals in their natal groups is acquired via associative learning at a young age and
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maintained through low-intensity, ritualized aggressive behaviors [4]. Female spotted hyenas are
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the more aggressive sex, and are socially dominant to all adult males not born in the clan.
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Females are philopatric, whereas males typically disperse from their natal clans between 2 to 5
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years of age [5,6]. Males may take several months to become successfully integrated into a new
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clan; immigrant males are subordinate to all natal animals and to immigrant males with longer
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tenure in the new clan [5,7].
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Data collection.
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The habitat occupied by all 5 clans was primarily open, rolling grassland with permanent water
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and year-round prey availability. Local prey density was calculated as the mean number of
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ungulates counted within 100 meters of multiple 4-km transect lines run through each territory at
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biweekly intervals for the 2 years prior to collection of each blood sample. Local prey density
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was available for only 1 year prior to sample collection for one high-ranking female from the Fig
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Tree clan and two high-ranking females from the Mara River clan. Per capita prey abundance
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was calculated as mean local prey density divided by the mean number of adults present within
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the respective clan during the 2 years prior to collection of each blood sample. Adults included
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all immigrant males, natal males older than 3 years, and females who were at least 3 years of age
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or who had borne their first litter, whichever came first. Within each clan, dominance rank
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matrices were constructed based on outcomes of dyadic agonistic interactions [2]. We
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standardized dominance rank to account for variation in clan size at time of darting; the
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individual with a relative rank of 1 was the highest-ranking animal in the clan, and the individual
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with a relative rank of -1 was the lowest-ranking. Except within matrilines, natal clan-mates are
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no more closely related than are immigrant male hyenas, which originate in many different clans
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[8]. Thus, among both natal females from different matrilines and male immigrants, relatedness
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is effectively zero [8]. It is rare for males to remain within their natal clans long after sexual
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maturity, so we were only able to obtain four telomere samples from three adult natal males.
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Hyenas were immobilized using Telazol (Zoetis, South Bend, IN; 6.5 mg/kg) administered via a
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lightweight plastic dart fired from a CO2-powered rifle (Telinject Inc., Saugus, CA). Blood
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samples were collected from the jugular vein into EDTA-treated vacutainers and promptly frozen
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in liquid nitrogen until they could be shipped on dry ice to Michigan State University where they
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were stored at -80°C.
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Telomere analysis: Genomic DNA was extracted using DNeasy Blood and Tissue Kits (Qiagen,
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Valencia, CA) and kept frozen at -80°C until telomere analysis. Once shipped on dry ice to
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Bucknell University, DNA was digested overnight with HinfI and RsaI, and separated using
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pulsed field gel electrophoresis (3 V cm-1, 0.5-7.0 s switch times, 14°C) for 21 hours, followed
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by in-gel hybridization at 37°C overnight with a radioactive-labelled telomere-specific oligo
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(CCCTAA)4. Gels were then placed on a phosphorscreen (Amersham Biosciences,
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Buckinghamshire, UK), which was scanned on a Storm 540 Variable Mode Imager (Amersham
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Biosciences). Position and strength of the radioactive signal were determined by densitometry
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using IMAGEQUANT 5.03v and IMAGEJ 1.42q. Mean telomere length was calculated using
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the formula: L = Σ(ODi x Li)/ Σ(ODi) where ODi is the densitometry output at position i and Li is
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the length (in basepairs) of the DNA at position i. For all samples, DNA integrity was evaluated
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empirically by a nucleic acid stain following agarose gel electrophoresis. DNA was deemed
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suitable for TRF length analysis if it was characterized as “a single compact crown-shaped band
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that migrates in parallel with the other samples on the gel” [9]. Two control hyena samples were
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run twice in each gel to determine intra- (2.8%) and inter-assay variability (3.2%).
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References
1. Holekamp KE, Smith JE, Strelioff CC, Van Horn RC, Watts HE. 2012. Society,
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demography and genetic structure in the spotted hyena. Mol. Ecol. 21, 613–632. (doi:
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10.1111/j.1365-294X.2011.05240.x)
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2. Frank LG. 1986. Social organization of the spotted hyaena (Crocuta crocuta). II.
Dominance and reproduction. Anim. Behav, 34, 1510–1527.
3. Kruuk H. 1972. The spotted hyena: a study of predation and social behaviour. Chicago,
IL: University of Chicago Press.
4. Smale L, Frank LG, Holekamp KE. 1993. Ontogeny of dominance in free-living spotted
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hyaenas: juvenile rank relations with adult females and immigrant males. Anim. Behav.
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46, 467–477.
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5. Holekamp KE, Smale L. 1998. Dispersal status influences hormones and behavior in the
male spotted hyena. Horm. Behav. 33, 205–216. (doi: 10.1006/hbeh.1998.1450)
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6. Van Horn RC, McElhinny TL, Holekamp KE. 2003. Age estimation and dispersal in the
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spotted hyena (Crocuta crocuta). J Mammal. 84, 1019–1030. (doi: 10.1644/BBa-023)
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7. East, ML, Hofer H. 2001. Male spotted hyenas (Crocuta crocuta) queue for status in
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social groups dominated by females. Behav. Ecol. 12, 558–568. (doi:
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10.1093/beheco/12.5.558)
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8. Van Horn RC, Engh AL, Scribner KT, Funk SM, Holekamp KE. 2004. Behavioral
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structuring of relatedness in the spotted hyena (Crocuta crocuta) suggests direct fitness
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benefits of clan-level cooperation. Mol. Ecol. 13, 449-458.
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9. Kimura M, Stone RC, Hunt SC, Skurnick J, Lu X, Cao X, Harley CB, Aviv A. 2010.
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Measurement of telomere length by the Southern blot analysis of terminal restriction
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fragment lengths. Nat. Protoc. 5, 1596–1607. (doi: doi:10.1038/nprot.2010.124)
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Table S1. Information on repeated samples from the same individuals.
1.
2.
3.
4.
5.
6.
7.
8.
9.
Hyena ID
Clan
Age1 (yr)
ali
eco
guci
mrph
bail
hex
nav
ldv
oak
Talek
Mara River
Talek
Talek
Talek
Talek
Talek
Talek
Talek
3.93
5.02
3.43
2.72
7.91
4.29
6.45
3.50
2.52
Age2 (yr)
9.24
5.32
6.51
3.72
11.28
7.42
9.71
7.57
7.84
Age3 (yr)
13.04
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Table S2. Information on the demography and territories of sampled clans.
Talek
Serena
North
Mara River
Serena
South
Fig Tree
years sampled
1999-2013
2012-2013
2002-2009
2012-2013
2006-2009
mean territory
size
57.3 km2
50 km2
31 km2
28 km2
52.91 km2
mean prey
density per
adult hyena
2.46
animals/
hyena
6.48
animals/
hyena
5.22
animals/
hyena
4.23
animals/
hyena
4.88
animals/
hyena
Table S3. Results from a simple linear regression of offspring adult telomere length on maternal
telomere length (df = 7).
intercept
maternal telomere length
estimate
11.71
0.17
s.e.
2.82
0.19
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t-value
4.16
0.93
p-value
0.004
0.39
R2
0.11