DOI: 10.5061/dryad.g1jwstr1f
title: Sexual selection matters in genetic rescue, but productivity benefits fade over time: A multi-generation experiment to inform conservation

description: Globally, many species are threatened by population decline because of
 anthropogenic changes leading to population fragmentation, genetic
 isolation, and inbreeding depression. Genetic rescue, the controlled
 introduction of genetic variation, is a method used to relieve such
 effects in small populations. However, without understanding how the
 characteristics of rescuers impact rescue attempts interventions run the
 risk of being sub-optimal, or even counterproductive. We use the Red Flour
 Beetle (Tribolium castaneum) to test the impact of rescuer sex, and sexual
 selection background, on population productivity. We record the impact of
 genetic rescue on population productivity in 24 and 36 replicated
 populations for ten generations following intervention. We find little or
 no impact of rescuer sex on the efficacy of rescue but show that a
 background of elevated sexual selection makes individuals more effective
 rescuers. In both experiments, rescue effects diminish 6-10 generations
 after the rescue. Our results confirm that the efficacy of genetic rescue
 can be influenced by characteristics of the rescuers and that the level of
 sexual selection in the rescuing population is an important factor. We
 show that any increase in fitness associated with rescue may last for a
 limited number of generations, suggesting implications for conservation
 policy and practice. 
description: <em>Husbandry</em> <em>T.
 castaneum</em> were kept in a controlled environment at 30°C and 60%
 humidity with a 12:12 light-dark cycle. Populations were kept on standard
 fodder consisting of 90% organic white flour, 10% brewer’s yeast and a
 layer of oats for traction unless otherwise stated. During the husbandry
 cycle, 2mm and 850µm sieves were used to remove pupae and adults from
 fodder. The following cycle was started by a set number of adults (line
 dependent, see below) being placed into containers with fresh standard
 fodder. The oviposition phase: populations were given seven days to mate
 and lay eggs before adults were removed by sieving to prevent overlapping
 generations. The fodder containing eggs was returned to the container. The
 development phase: eggs were kept in the containers for 35 days to allow
 the eggs to develop into mature adults. Around day 21 of the development
 phase, pupae were collected to obtain known-sex virgin individuals which
 were then used to start the next generation. The pupae were kept as
 virgins in single-sex groups of 20 for 10 days to allow them to complete
 development. Once mature, the cycle began again with those beetles going
 into fresh fodder to form a population of males and females.
 <em>Tribolium castaneum</em> lines
 Krakow Super Strain (KSS):
 was created by mixing fourteen laboratory strains to maximise genetic
 diversity in a single strain (Laskowski et al., 2015). This was used as
 the outbred treatment in the genetic rescue experiments.
 Inbred Lines: Founded from KSS and inbred
 through three single-pair bottlenecks in the first, fifth and seventh
 generations. Between bottlenecks, the lines were maintained at a maximum
 population size of 100 randomly selected adults. Of the initial 30 lines,
 24 survived the inbreeding treatment and 12 lines were maintained and used
 for experiments. Sexual Selection
 Lines: polyandrous and monogamous lines were created from the
 Georgia 1 stock (Haliscak and Beeman, 1983; Lumley et al., 2015). Each
 polyandrous line (n=3) was maintained each generation in twelve groups
 each consisting of five males and one female. Following oviposition, the
 eggs from all groups in a line are mixed to form one population from which
 the next generation’s groups will be sourced. For each monogamous line
 (n=3) twenty separate mating pairs are bred. Following oviposition, the
 eggs from all pairs are mixed and the next pairs are sourced from this
 population to maintain that line. The number of groups and pairs in each
 regime results in a theoretical Ne = 40 in each treatment (Godwin et al.,
 2020). These regimes had been maintained for 150 generations when rescuers
 were taken. The polyandrous lines are hereafter referred to as sexual
 selection lines, and monogamous as no sexual selection.
 <em>Genetic rescue protocol</em>
 Replicate experimental inbred populations were created from the
 inbred lines to serve as populations to be rescued. Pupae were sexed and
 placed into plastic dishes with lids, containing 10ml standard fodder in
 single-sex groups. 10±2 days after eclosion, ten males and ten females
 from a given line were placed in a 125ml tub with 70ml of standard fodder
 creating populations each containing twenty adult beetles at a 1:1 sex
 ratio for the oviposition phase. On day 20±1 of the development phase,
 pupae were again taken from the populations using the method outlined
 above to create the next non-overlapping generation.
 Populations were maintained using twenty reproducing adults per
 generation, not allowing population growth. This allowed us to maintain a
 roughly constant population density during offspring development across
 generations, avoiding the confounding influence of negative
 density-dependence on offspring production (Duval et al., 1939; King and
 Dawson, 1972; Janus, 1989). Each experimental
 population was randomly assigned an ID number, to avoid bias when
 handling. After being established at the experimental size, the
 populations were maintained in experimental conditions for one generation
 to avoid transgenerational density effects affecting the genetic rescue
 results (Đukić et al., 2021). The rescue treatments were applied in the
 second generation under experimental conditions. In each population, a
 single beetle was replaced with a rescuer thus maintaining the 1:1 sex
 ratio and population size, avoiding any increase in productivity due to a
 demographic rescue. Rescuers taken from their source populations as pupae
 were age-matched as closely as possible to individuals in experimental
 populations. On day 37 of the development phase experimental populations
 were frozen at -6°C and mature offspring were counted as a measure of
 productivity (our metric for population fitness). If a population was
 removed from the experiment because of slow development (pupae were not
 available to establish the next generation), that population was analysed
 as part of all generations prior but excluded henceforth.
 <em>The sex of the rescuer in genetic
 rescue</em> Due to logistic issues with
 ventilation, four out of the 12 experimental inbred populations failed to
 produce offspring in generation 0. From each of the remaining eight inbred
 lines, three replicate populations were created and assigned to one of
 three treatments; No Rescue control (ten inbred line males, ten inbred
 line females); Male Rescue (nine inbred line males, one KSS male, ten
 inbred line females); and Female Rescue (ten inbred line males, nine
 inbred line females, one KSS female; Figure 1). Populations were
 maintained for ten, non-overlapping generations.
 <em>Sexual selection and genetic
 rescue</em> We investigated the impact of a
 rescuer’s sexual selection history on the effectiveness of genetic rescue.
 From 12 inbred lines, three replicate populations were created and
 assigned to one of three treatments; No Rescue Control (ten inbred line
 males, ten inbred line females); Sexual Selection Rescue (nine inbred line
 males, one polyandrous male and, ten inbred line females); No Sexual
 Selection Rescue (nine inbred line males, one monogamous male, ten inbred
 line females; Figure 2). A single polyandrous and single monandrous line
 were used as the source for rescuers. Populations were maintained for nine
 generations. <em>Stressful
 conditions</em> To test rescue under stress
 conditions, duplicate rescue populations were established from each
 rescued line at generation five in the ‘sex’ experiment, and generation
 six in the ‘sexual selection’ experiment. These were maintained as in the
 main experiments (until generation ten and nine respectively), but with a
 reduction in the yeast content of the fodder, which is the main source of
 protein for the experimental populations. This reduction generates
 nutrient stress in <em>T. castaneum</em> (Godwin et al.,
 2020). In the ‘sex’ experiment fodder contained 0% yeast and 1% yeast in
 the ‘sexual selection’ experiment (because of low survival with zero
 yeast). <em>Statistical
 analyses</em> Statistical analyses were carried
 out in R V4.4.1 (R Core Team, 2024) utilising R studio version
 2024.04.2+764 (Posit team, 2024). Tidyverse (Wickham et al., 2019), stats
 (R Core Team, 2024), Rmisc (Hope, 2022)and googlesheets4 (Bryan, 2023)
 were used for data management and exploration. Plots were created using
 ggplot2 (Wickham, 2016). The distribution of data was checked using the
 shapiro.test function (R Core Team, 2024). Generalised Linear Mixed Models
 (GLMMs) were fitted to test for differences in productivity between the
 experimental treatments using glmmTMB (Brooks et al., 2017). Model fit was
 checked using DHARMa (Hartig, 2022). Model parameters were checked for
 collinearity using variance inflation factor (Vif) scores with the
 check_collinearity function from performance (Lüdecke et al., 2021). There
 were no issues with overdispersion or collinearity (VIF: &lt;3 for all
 variables) in any models. R<sup>2 </sup>was determined using
 the r.squaredGLMM function in MuMIn (Bartoń, 2024). Post-hoc pairwise
 Tukey tests were carried out using multcomp (Hothorn et al.,
 2008). Within each experiment, we fitted GLMMs with the
 same model structure, using a negative binomial distribution to model
 productivity counts, which provided better model fit than a Poisson
 distribution. Productivity was the response variable, with treatment,
 generation and generation<sup>2</sup> as fixed effects. Inbred
 line of origin and experimental population ID were included as random
 effects, with ID nested within inbred line. Interaction terms (treatment x
 generation, treatment x generation<sup>2</sup>) were initially
 included but removed from the model if not significant. The
 generation<sup>2</sup> factor was not significant in the
 models for populations under stressful conditions and was therefore
 removed. When a quadratic effect of generation was found in a model, we
 then tested to see if this was due to an increase and a decrease in
 productivity, rather than just one of these. We did this using two
 separate GLMMs (with the same factors as previously) run on the data split
 into generations 1-5 and 5-10. These GLMMs were fitted with treatment and
 generation as fixed effects, ID nested within inbred line as a random
 effect. GLMMs were also fitted on generations 2 and 3 individually (Table
 S3 + S4) in the ‘sex’ experiment, these single-generation models used a
 Poisson distribution, productivity as a response variable and treatment as
 a fixed effect. Random effects were the same as above. This was to test at
 which point the rescue treatments resulted in a significant difference
 from the control, to see if there were differences in the speed of male or
 female rescue.
description: # Sexual selection matters in genetic rescue, but productivity benefits
 fade over time: A multi-generation experiment to inform conservation
 [https://doi.org/10.5061/dryad.g1jwstr1f](https://doi.org/10.5061/dryad.g1jwstr1f) ## Description of the data and file structure Test of sex and, sexual selection background, of genetic rescuers on the fitness of an inbred population. Null entries in the data sets were not included in that or any future generations. ### Abbreviations KSS - Krakow Super Strain, Outbred ancestral population. C - Control treatment, Inbred populations that received no rescue. F - Female rescue treatment, Inbred populations that received rescue by a female KSS. M - Male rescue treatment, Inbred populations that received rescue by a male KSS. Control - Control treatment, Inbred populations that received no rescue. Mo - Monogamy rescue treatment, Inbred populations that received rescue by a monogamous beetle. Po - Polyandry rescue treatment, Inbred populations that received rescue by a Polyandrous beetle. Mo control - Control population of monogamous beetles in experimental setup conditions (Not included in the analysis). Po control - Control population of polygamous beetles in experimental setup conditions (Not included in the analysis). ### Files and variables #### File: SEL-GR.csv **Description:** Productivity data for experiment with genetic rescue by different sexual selection regimes ##### Variables * ID: Identifying number * Generation: Generation after rescue * Productivity: Number of adult offspring produced * Isoline: Inbred line Identification * Treatment: Treatment applied #### File: SS-GR.csv **Description:** Productivity data for experiment with genetic rescue by different sexes ##### Variables * ID: Identifying number * Generation: Generation after rescue * Productivity: Number of adult offspring produced * Isoline: Inbred line Identification * Treatment: Treatment applied #### File: Stress-SEL-GR.csv **Description:** Productivity data for experiment with genetic rescue by different sexual selection regimes in stressful conditions ##### Variables * ID: Identifying number * Generation: Generation after rescue * Productivity: Number of adult offspring produced * Isoline: Inbred line Identification * Treatment: Treatment applied #### File: Stress-SS-GR.csv **Description:** Productivity data for experiment with genetic rescue by different sexes in stressful conditions ##### Variables * ID: Identifying number * Generation: Generation after rescue * Productivity: Number of adult offspring produced * Isoline: Inbred line Identification * Treatment: Treatment applied ## Code/software Statistical analyses were carried out in R V4.4.1 (R Core Team, 2024) utilising R studio version 2024.04.2+764 (Posit team, 2024). Tidyverse (Wickham et al., 2019), stats (R Core Team, 2024), Rmisc (Hope, 2022)and googlesheets4 (Bryan, 2023) were used for data management and exploration. Plots were created using ggplot2 (Wickham, 2016). The distribution of data was checked using the shapiro.test function (R Core Team, 2024). Generalised Linear Mixed Models (GLMMs) were fitted to test for differences in productivity between the experimental treatments using glmmTMB (Brooks et al., 2017). Model fit was checked using DHARMa (Hartig, 2022). Model parameters were checked for collinearity using variance inflation factor (Vif) scores with the check_collinearity function from performance (Lüdecke et al., 2021). There were no issues with overdispersion or collinearity (VIF: &lt;3 for all variables) in any models. R2 was determined using the r.squaredGLMM function in MuMIn (Bartoń, 2024). Post-hoc pairwise Tukey tests were carried out using multcomp (Hothorn et al., 2008). full_script - R script as one whole file. SEL-GR LOAD - Load packages and data for sexual selection experiments SEL-GR FIGURE - Create figure for sexual selection experiments SEL-GR MODELS - Run models for sexual selection experiments SS-GR LOAD - Load packages and data for sex experiments SS-GR FIGURE - Create figure for sex experiments SS-GR MODELS - Run models for sex experiments ## Supplementary files README.pdf - PDF version of the README Figure 1 - Figure 1 from the manuscript Figure 2 - Figure 2 from the manuscript Figure 3 - Figure 3 from the manuscript Figure 4 - Figure 4 from the manuscript