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Supplementary Information
Post-mating sexual selection increases lifetime fitness
of polyandrous females in the wild
Diana O. Fisher, Michael C. Double, Simon P. Blomberg, Michael D. Jennions, Andrew
Cockburn
Supplementary methods and discussion
Genotyping. We used an ammonium acetate extraction procedure 26 to obtain DNA
from ear tissue of mothers and potential sires or toe-buds from polyandrous females’
offspring. Mothers and potential sires were genotyped at five loci and offspring at 2-5
loci that were potentially informative about paternity 23. PCR products were labeled
using M13-labelling 31 and run on a 3100 capillary automated sequencer (Applied
Biosystems). PCR conditions were identical for all loci: 3 min at 94 oC; 25 cycles of 30
s at 94 oC, 30s at 55 oC, 30s at 72 oC; 10 cycles of 30s at 94 oC, 30s at 50 oC, 30s at 72
C; 5 min at 72 oC. Males that possessed all the offspring’s paternal alleles were
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assigned paternity. Each offspring matched the genotype of only one potential sire.
Offspring sex and mating treatment. Analyses were run in Genstat 8.0 29 or R2.3.128.
GLMM generalized linear mixed models were fitted using the Matrix package for R27.
To test for factors affecting the birth sex ratio we ran a generalized linear model with
binomial error and the proportion of each litter that was male as the dependent variable
(numerator: number of males; denominator: litter size). Year and mating treatment were
fixed factors and block was a random factor. There was no effect of mating treatment
(2= 0.27, P = 0.60) or year (2= 0.36, P = 0.55) on the birth sex ratio, nor was there
any interaction between year and mating treatment (2= 0.31, P = 0.58). To test
whether offspring sex affected survival to weaning we used a generalised linear mixed
model with binomial error and offspring nested within litter. To be conservative we only
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used data from 2004 where survival is known for both sexes with complete certainty.
Sex and mating treatment were fixed factors and block was a random factor. There was
no effect of sex on offspring survival (2= 1.1, P = 0.30). More importantly, the effect
of mating treatment on survival was the same for sons and daughters (2= 0.26, P =
0.61).
Separate and combined analyses of the effect of mating treatment. In the main text
we presented separate analyses for each experiment to show that the main effect of
mating treatment on offspring survival to weaning was significant in both years. If the
data from the two experiments are combined the positive effect of polyandry on
offspring survival to weaning is very clear (2= 43.4, P < 0.0001). There is no
interaction between mating treatment and experimental year (2= 0.4, P = 0.53). When
combining the two experiments, the proportion of females failing to produce sufficient
young to attach to all their teats did not differ between years (2= 0.1, P = 0.75) and
was higher for monandrous females (2= 6.0, P = 0.014). The effect of mating
treatment did not differ between years (2= 2.5, P = 0.11), however, the effect was only
significant in 2003 and there was no effect in 2004 (P=0.46, see main text). Unlike the
effect of polyandry on survival, its effect on the initial production on offspring was
therefore not highly replicable between years.
Analysis of the number of young attached at birth. We tested the effects of treatment
and sire ejaculate competitiveness on the proportion of females that had young attached
to all their teats (binomial data), rather than the number of young attached at birth. We
did so because the maximum number of young that a female antechinus can raise is
constrained by the number of teats, and many females have complete litters.
Additionally, two females in our experiments had 9 or 10 teats.
Analysis of effect of ejaculate competitiveness on offspring survival. To test for the
effect of sire ejaculate competitiveness on offspring survival we only used offspring
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from monandrous females. In principle, we could have used offspring of known
paternity from polyandrous females as well. We could not do so, however, because the
dependent variable in the analysis was the proportion of offspring per litter surviving to
weaning. Offspring from the same litter cannot be treated as statistically independent
data. Due to the low paternity of less competitive males mated to polyandrous females,
this would have resulted in a highly unbalanced data set (i.e. strong skew towards an
over-representation of competitive males’ offspring born to polyandrous females) as
well as proportions where the denominator would have taken on very low values (i.e.
the total number of offspring per litter sired by a male with low paternity was often 0 or
1).
The effect of mating treatment on offspring growth. Despite the tendency of
polyandrous females to give birth to slightly more offspring than monandrous females,
this did not decrease the growth rate of their young. Analyses were conducted with a
linear mixed model using REML to estimate parameters. Comparisons of models with
different fixed factors were performed using maximum likelihood (ML). Mating
treatment and year were fixed factors and litter identity and block were treated as
random factors. We tested for a mating treatment effect prior to most offspring
mortality, and measured the body length of young when they were 31-32 days old. The
young of polyandrous mothers were significantly longer (2= 6.2, P = 0.01;
polyandrous: 13.1 ± 0.07 mm; monandrous: 12.8 ± 0.06 mm), and there was no
significant difference in body length between years (2= 3.1, P = 0.08). There was no
difference in the effect of mating treatment between years (2= 2.3, P =0.13). Although
the difference in body size is small, it confirms results from the closely related species,
Antechinus agilis, where experimental manipulation of the number of mates also led to
polyandrous females producing slightly, but significantly, larger offspring than
monogamous females 23.
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Relatedness between mates. The combination of female philopatry (mothers and
daughters, and sisters share nests and foraging ranges), male natal dispersal 20, the death
of all fathers 18,21, and the fact that the study site was part of a large interconnected
population meant that matings between close relatives were extremely unlikely in 2003.
In 2004, mating between kin was explicitly prevented. The site of origin of nine of the
males was known and each was paired with a female born in a separate forest fragment,
thus preventing any sibling-sibling, mother-son, cousin or grandmother-grandson
matings. The other males were not caught during comprehensive trapping of young in
2003, so they must have been unrelated immigrants.
Reproductive biology of male antechinuses. Carnivorous marsupials in the family
Dasyuridae include semelparous species (e.g. the genus Antechinus) and iteroparous
species 32. Species with both types of life history ejaculate small numbers (105-106) of
very large sperm, which move particularly efficiently, so more of them reach the top of
the oviduct than in similar-sized rodents that ejaculate orders of magnitude more sperm
33,34
. Female dasyurids can store sperm in specialised crypts in the isthmus of the
oviduct for two weeks, allowing copulation and conception to be decoupled 35. In
genera with obligate male death after mating, including Antechinus, spermatogenesis
fails before the mating season, so males begin with a finite number of stored sperm 36.
Even if they fail to mate, these sperm are lost through urination, so males are sterile at
the end of their first mating season. Testicular failure seems likely to be a consequence
of obligate male death. It might occur because there is no selection for late-acting alleles
that allow spermatogenesis, or there is active selection against spermatogenesis because
of its cost, or against testicular function because it impairs regulation of the hormonal
pathways that enable antechinuses to use gluconeogenic mobilisation of body reserves
to sustain them during the three week mating season, when they appear to severely
reduce foraging 37. Male antechinuses generally copulate for five to 14 consecutive
hours with each female and ejaculate around three hours after mating starts 38,39. The
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strength of our results may be due in part to this unusual life history, and the effect of
the extreme physiological stress associated with intense sperm competition in males.
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marsupials. In: Archer M (ed) Carnivorous Marsupials, vol I. Royal Zoological Society
of NSW, Sydney, pp 1-11.
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spermatozoa from the dasyurid marsupial, Antechinus stuartii. Reprod. Fertil. Dev
2:107-114.
34. Taggart DA, Temple-Smith PD (1990b) Effect of breeding season and mating on
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mouse, Antechinus stuartii. J. Reprod. Fertil. 88:81-91.
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from marsupials? Proc. R. Soc. Lond. 221, 221-233 (1984).
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Mouse Antechinus-Stuartii Macleay (Dasyuridae, Marsupialia). Aust. J. Zool. 31:445466.
37. Cockburn A (1992) Evolutionary ecology of the immune system: why does the
thymus involute? Funct. Ecol. 6:364-370.
38. Taggart, D. A. & Temple-Smith, P. D. Transport and storage of spermatozoa in the
female reproductive tract of the brown marsupial mouse, Antechinus stuartii
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39. Shimmin, G. A., Jones, M. Taggart, D. A., Temple-Smith, P.D. Sperm transport and
storage in the Agile Antechinus (Antechinus agilis). Biol. Rep. 60, 1353-1359 (1999).