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Electronic Supplementary Material (ESM)
Methods
Drosophila strains
The experiment harnessed five isofemale strains of D. melanogaster . Four of these were derived from wild populations spanning three continents: Dahomey (denoted as DAH, derived from a mass-bred population collected in 1970 from Benin, Africa); Puerto Mont
(PUER, Chile, isofemale derived from massbred [1]); Zimbabwe (denoted ZIM, Africa,
Zim53 from JWO Ballard [2])
; and Madang (MAD, New Guinea, derived from mass-bred
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stock [3]). A fifth strain (
w1118 ) was sourced from the Bloomington Stock Centre (stock
number 5905) and is isogenic (constructed by John Roote, Cambridge, UK, Canton-S) [4].
Each wild type strain was subjected to a heavy inbreeding protocol, via 12 generations of full-sibling (brother-sister) mating. In this way, four standardised near-isogenic genotypes
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All strains were treated using 0.164 mg mL-1 tetracycline [5] in each of two
generations, at least four generations prior to the commencement of this experiment to ensure that individuals were free of Wolbachia infection.
All fly strains were cultured at 25 ºC, on a 12:12 h light:dark cycle, in 10 dram vials on potato-dextrose-agar medium
, and at moderate larval densities (150 eggs per vial). We used anaesthesia under light carbon dioxide when collecting virgin flies and establishing crosses.
All transfers of flies across vials were conducted without CO2.
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Experimental design
The experiment was carried out in four blocks that were separated temporally over four sequential generations. The grandparents of the focal flies used in the experiment were maintained across numerous replicate vials per strain, with each replicate containing eight to
10 pairs provided with ad libitum access to dry live yeast. Larval densities were controlled by trimming the number of eggs to 150 per vial. Virgin males and females were collected from each vial 10 days after oviposition, and the following crossing scheme implemented to generate the focal males.
Virgin males of each wild type strain were either crossed to virgin females of the same strain (eight pairs of virgins per replicate), to create a standard homozygous focal male
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50 genotype per strain, or they were crossed to virgin females from w1118 (eight pairs per replicate) to create a standard heterozygous focal male genotype per strain in the F1 generation (Table S1). Our motivation here was to assay the four focal genotypes when they were expressed in their diploid (i.e. homozygotic) and haploid (i.e. heterozygotic) states. If we found convergence across homozygous and heterozygous lines in the genotypic effect on sperm viability across Genetic strains, this would indicate that the genotypic effects on the sperm viability phenotype were driven primarily by the contribution of alleles acting additively (i.e. additive genetic variance at autosomal loci). If the key allelic variance for sperm viability was additive, then divergence in sperm viability across genotypes should exhibit a similar pattern across the focal genotypes in both the homozygous and heterozygous states, although the absolute magnitude of change across genotypes would be larger when expressed in homozygous state (since in heterozygous state – the copy number associated with each focal genotype is halved and hence diluted). Furthermore, by assaying these genotypes in a homozygous state, we could unmask effects across genotypes that were driven by autosomal recessive alleles of large effect, by way of a Genetic Strain ( DAH, PUER, ZIM,
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MAD) by Genetic status (homozygote, heterozygote)
interaction. Finally, given that the homozygote and heterozygote states actually represent cases of extreme inbreeding (although note lethal recessives would have been purged during the line creation) and extreme outbreeding respectively, we would predict an overall heterosis effect (a general increase in mean sperm viability across all genotypes) for males whose genotypes were expressed in the heterozygous state.
F1 sons of the outbred crosses, thus, possessed one set of chromosomes 2 to 4 from
58 each parent (wild type strain and w1118 ), a Y chromosome from the respective wild type
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75 strain, and an X chromosome from w1118 . In each block, we generated three replicate crosses per wild type strain, in each of the homozygous and heterozygous combinations. Egg numbers per vial were trimmed back to 100. F1 virgin males were collected from these vials
10 days after ovipositioning, and stored in groups of eight, with each group of males then assigned to an age treatment consisting of two classes: young and old. Males assigned to the young class were held in vials with ad libitum access to live yeast for three days prior to their dissection. Males in the old class were held for 23 days prior to their dissection, and transferred to fresh vials with ad libitum yeast every third day.
Dissections
Individual males were anaesthetised on ice and dissected in a drop of HEPES-buffered saline (Modified Alsever's solution: 27 mM sodium citrate, 336 mM sodium chloride, 115 mM glucose, 9 mM EDTA; 20 mM HEPES, pH 7.0) on a glass slide. The male reproductive tract was removed and gently straightened. One of each pair of testes and both accessory glands (when intact) were photographed under 40 × magnification on a compound microscope. One seminal vesicle was isolated from the rest of the reproductive tract, transferred to a 2.5 μL drop of HEPES-buffered saline on a glass slide, and punctured to
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100 release the sperm. The glass slide with sperm was then stored in a dark humid chamber for exactly 2 h before commencing a sperm viability assay.
Sperm viability and trait measurements
Sperm viability was quantified as the proportion of live sperm in the total sperm sample using the LIVE/DEAD sperm viability kit (Molecular Probes, 22 μL of SYBR-14 and 44 μL
of propidium iodide per 1mL of HEPES-buffered saline, adapted from [6]). We added 1.25
μL of the fluorescent stain to the sample 5 min before it was scored. All of the live (stained green) and dead (stained red) sperm cells on the slide were counted at 200 × magnification, on a fluorescence microscope fitted with a UV filter. A small proportion of cells was doubly stained (i.e. both green and red) and these sperm were counted as dead. We also photographed the heads of three haphazardly selected live sperm cells per slide at 400 × magnification.
We measured testes, seminal vesicle and accessory glands areas, and sperm head length using the public domain program ImageJ v1.43u (http://rsb.info.nih.gov/ij/). Whenever possible, both accessory glands were measured and their area averaged.
Data analysis
We performed d ata exploration using a Cleveland dotplot to investigate potential outliers, checked for collinearity among covariates and non-linear relationships between explanatory variables. Our initial binomial GLM contained the following predictors of sperm viability: genetic status, genetic line, age, total sperm number, accessory gland area, seminal vesicle area, testis area, sperm length, and all two-way interactions among them. With the exception of sperm length, all other reproductive traits varied consistently across male ages
(Fig S1), and their inclusion alongside male age within the same model was not possible.
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We therefore fitted two sets of models.
First, we fitted a model in which Age (3,24
102 days), Genetic line ( DAH, PUER, ZIM, MAD) , and Genetic status (homozygote,
103 heterozygote) were included as fixed factors and sperm length as a covariate (centred around
104 0). We then fitted age-specific models (one for young males, one for old), in which all of the
105 reproductive morphological traits (each centred around 0) were fitted as covariates, alongside
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119 the fixed effects of Genetic line and Genetic status, and interactions between these were tested.
To deal with the detected overdispersion, we used a quasi-GLM model with an
‘empirical scale parameter’ of 300.06 to adjust the errors, as they were not binomial. Model
selection was done using F tests to assess which terms to drop in the quasi-GLM models [7]
.
M
odel validation followed [7]
, with graphical diagnostics for the assumptions of the model .
For each model, a full model, including first order interactions, was reduced by removing non-significant terms one parameter at a time using F-tests. For simplicity, only the final models are presented with Type II sum of squares.
Discussion
Previously, inbreeding has been shown to affect the expression of sperm traits [16, 17] and
120 male fertility outcomes [18, 19]. We found general effects of genome-wide heterozygosity
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122 levels on the expression of sperm viability, which implicate deleterious effects of inbreeding on sperm quality. We acknowledge the alternative possibility that the w
1118
strain, which
123 contributed a haploid copy of the nuclear genome to all heterozygous focal males, might have
124 affected the magnitude of this association rather than the heterozygosity per se . However, this
125 possibility seems less likely for two reasons. First, there was a clear absence of a genotype
126 (genetic strain) effect in this study, with four distinct wild-sourced genotypes, expressed in
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127 the homozygous (diploid) state, failing to show phenotypic divergence in the expression of
128 sperm viability (Genetic strain – see Table 1). Furthermore, there were no interactions
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130 involving Genetic strain and the other measured factors, on the sperm viability phenotype.
Second, the w
1118 genotype was only ever expressed in heterozygous (i.e. haploid) form – that
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132 is, invariably alongside each of the four wild type genotypes, and this should further dilute any main effect per se of the w
1118 genotype in driving these effects. On the balance of these
133 two points, it seems more plausible that the heterozygosity effects are driven by
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139 heterozygosity per se , and not by a confounding factor.
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Table S1. Crossing scheme used to generate focal F1 males.
Genetic Status Age
Focal male genotype
(paternal : maternal contribution)
Young
Old
DAH : DAH
Homozygous
Young
Old
Young
Old
Young
Old
Young
PUER : PUER
ZIM : ZIM
MAD : MAD
DAH : w1118
Old
Young
Old
Heterozygous
PUER : w1118
Young
Old
Young
Old
ZIM : w1118
MAD : w1118
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4000
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1000 a a
0.2
0.16
0.12
0.08
0.04
b b
0.2
0.16
0.12
0.08
0.04
0.05
0.04
0.03
0.02
0.01
d d
0
Young Old
0
Young Old
Male age
0
Young Old
0
Young Old
Fig S1.
Mean  S.E. a) sperm number, b) testes area, c) accessory gland area and d) seminal vesicle area for young and old males.
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ESM References
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