Stock culture maintenance

The red flour beetle Tribolium castaneum is a tractable research model for studying reproduction29,64,65. We used the outbred ‘Kraków Super Strain’ (KSS) created in 2008 by combining 35–60 individuals from 11 different strains to promote genetic diversity66. Stocks were maintained under standard conditions (30 ± 1 °C, 60 ± 5% RH and 16L: 8D photoperiod) in ad libitum fodder consisting of organic flour and yeast (9:1 by volume) topped with oats for traction65. Populations were maintained as non-overlapping generations, renewed every 35 days by transferring ~300 sexually mature adults to fresh fodder for 7 days mating and oviposition, then removing adults to allow egg and larval development. Unless otherwise stated, all individuals used in experiments were sexed as pupae, kept in single-sex groups of 20 individuals in 5 cm petri dishes to eclosion and sexual maturity at 12 ± 2 days, then randomly assigned to treatments. During maturation, one sex was identified with a dot on the dorsal thorax using correction fluid (Tippex, France). This marking method has no significant effect on reproductive output across 20 days of oviposition (marked versus unmarked females χ2 (1,36) = 0.7, P = 0.407; z = −0.8, P = 0.407, n = 19 + 19).

Heatwave conditions

Heatwave treatments exposed individuals for 5 days to temperatures that exceeded the optimum by 5 °C, corresponding with the common definition of a heatwave event14. The optimum temperature for population productivity in T. castaneum is 35 °C29,67, which our assays confirmed (Fig. 1). Experimental heatwaves therefore exposed individuals to temperatures of 40 to 42 ± 1 °C. These conditions have been recorded in the natural environment across more than 90 countries68. Heatwave conditions were applied using Octagon 20 incubators (Brinsea Ltd, UK), and the humidity of all treatments was maintained at 60 ± 5% RH. Beetles were exposed to heatwaves in single-sex groups of 20 individuals in 5 cm petri dishes containing standard fodder and positioned in the central plane of the incubator. Temperatures did not exceed 1 °C above or below the treatment set point, checked using a 35–45 °C mercury incubation thermometer (G.H. Zeal Ltd, Zeal House, 8 Deer Park Road, London, SW19 3UU, U.K.) calibrated to United Kingdom Accredited Service standards (Charnwood Instrumentation Services Ltd, 81 Park Road, Coalville, Leicestershire, LE67 3AF, UK). Following treatments, all individuals experienced 30 ± 1 °C for 24 h, before running reproductive output assays at 30 ± 1 °C.

Reproductive output: heatwave impacts on adults

Supplementary Figure 4a presents these experimental protocols. Reproductively mature males and females were exposed to 5-day thermal treatments at 30 °C (n Males = 79, n Females = 75), 35 °C (n M = 33, n F = 34), 38 °C (n M = 48, n F = 43), 39 °C (n M = 43, n F = 35), 40 °C (n M = 48, n F = 35), or 42 °C (n M = 42, n F = 28). After treatment, and a further 24 h at 30 °C, they were monogamously paired with untreated mates for 2 days at 30 °C in 4 ml vials containing 0.5 g flour and yeast topped with oats. Following mating, males were removed and females isolated in 5 cm petri dishes for oviposition into 7 g flour and yeast with 3 g of oats on the surface for 20 days at 30 °C, using two separate 10-day blocks to reduce overlapping generations (Supplementary Figure 4a). After removing the female at day 20, eggs and larvae produced over this period were left to develop in standard conditions at 30 °C for 35 days until they emerged to be counted as mature adults. Reproductive output of each breeding pair was therefore the number of offspring successfully produced over 20 days of oviposition, which correlates significantly with lifetime output and accounts for ~50% of a female’s total potential reproductive output under similar conditions across 150 days of oviposition66.

Reproductive output: heatwave impacts on sperm in females

Supplementary Figure 4b presents these experimental protocols. Impacts on individual spermatozoa were measured by exposing sperm stored within the reproductive tract of mated females to heatwave conditions, comparing against females which received the same heatwave treatment but immediately prior to mating and sperm storage (Supplementary Figure 4b). Thus, females were either mated, then exposed to heatwaves (n = 62); or exposed to heatwaves, then mated (n = 55). Following either treatment, females were transferred to 5 cm petri dishes for oviposition across three separate 5-day blocks under standard conditions, counting the number of offspring produced after 35 days of development. Five-day blocks were applied so that we could control for any differential sperm ageing effects that may have occurred between insemination and the period of reproductive fitness measurement: reproductive output by females in the ‘sperm + female heated’ treatment was compared across the first 10 days of oviposition following treatment (and therefore 5 days following the timing of insemination), whereas output in the ‘unmated female heated’ was compared following oviposition from day 5 to 15 (again, 5 days following insemination). We also ran comparisons of reproductive output for all 15 days of oviposition. Both comparisons showed significant 26 to 31% declines in female reproductive output when females had been exposed to heatwave conditions containing sperm in storage. Results controlling for sperm age and comparing reproductive output across 10 days of oviposition are in the main document and Fig. 2a. Comparisons of the total 15 days of reproductive output yielded similar results with significant declines in reproductive output when sperm had experienced heatwave conditions within female storage (χ2 (1,115) = 17.1, P < 0.001; z = −4.1, P < 0.001).

Reproductive output: heatwave impacts on sperm competition

Supplementary Figure 5 presents these experimental protocols. To assess impacts within the relevant context of sperm competition, we measured how heatwave conditions influenced a male’s subsequent ability to win fertilisations within females that had previously been mated to untreated, marker males. Males were sexed as pupae, and then isolated from eclosion until experimental mating to standardise and prevent any confounds from uncontrolled same-sex behaviour activity69. Treatment males were exposed to 5 days at 30 °C (controls) or 42 °C (heatwaves), followed by 24 h at 30 °C. During this 24 h period, control females were mated to ‘Reindeer’ marker males. The Reindeer (Rd) mutation for clubbed antennae is dominant and maintained homozygous in our stock. Offspring sired by Reindeer males will inherit the clubbed antennae phenotype, whereas offspring sired by the wild type males develop normal filiform antennae, allowing paternity to be assigned treatment group males70. After 24 h mating with Rd males, females were then mated with either control (n = 65) or heatwave-treated (n = 51) males for 24 h, and then transferred to oviposit individually in 5 cm petri dishes for 7 days. Following oviposition, offspring were left to develop for 35 days, after which the relative numbers of wild type and Rd offspring were counted to measure differences in paternity and relative sperm competitiveness between the heatwave and control male treatment groups.

Reproductive output: double heatwave impacts

Supplementary Figure 6 presents these experimental protocols. To measure the impact of additional heatwaves, adult males were exposed to three treatments: (1) Control: 5 days of exposure to 30 °C (n = 20); (2) Single heatwave: 5 days of heatwave exposure at 42 °C (n = 35); and (3) Double heatwaves: 5 days of heatwave exposure at 42 °C followed by 10 days at 30 °C followed by a second 5 days of heatwave exposure at 42 °C (n = 29). Following each treatment, males were maintained for 24 h at 30 °C before being monogamously paired to untreated adult mature females for 2 days in 4 ml vials, after which females were transferred individually to 5 cm petri dishes for 20 days of oviposition in standard conditions, across two 10-day blocks. After 20 days, females were removed and all offspring allowed to develop for 35 days so that offspring production could be counted. To minimise developmental effects through initial spermatogenesis, all males were reproductively mature (12 ± 2 days post eclosion) and received their initial 5-day treatments simultaneously, with males in group 3 experiencing their second heatwave at age 27 ± 2 days post eclosion. Thus, all males were reproductively mature when exposed to single or double heatwaves (Supplementary Figure 6).

Heatwave impacts on male mating behaviour

Males sexed as pupae were individually isolated before their mating behaviour assay to prevent any same-sex activity and to standardise all individuals prior to each trial64,69. At adult maturity, males were exposed for 5-day treatments at 30 °C (n = 25), 39 °C (n = 24), 40 °C (n = 21), 41 °C (n = 24) or 42 °C (n = 14), followed by 24 h at 30 °C (Supplementary Figure 4c). Following treatment, males were paired with untreated control females at 30 °C in 1 cm2 mating arenas for 1 h, and all mating activity video-recorded using Sony digital video cameras. Replaying the 1-h film sequence for each pair, we recorded: (1) the period of latency to first mating, (2) the total number of matings and (3) the duration of each mating. Matings were defined when the pair achieved unbroken mounting and copulatory contact for more than 35 s, which is the average minimum time for successful spermatophore transfer in T. castaneum71.

To assess the probability of subsequent spermatophore transfer in matings by males previously experiencing heatwave conditions, we ran an additional assay in which males (n = 36) that had previously received a 5-day 42 °C treatment were paired monogamously with untreated females for 1 h in 1 cm2 mating arenas, after which females were frozen at −20 °C, before being dissected to check for successful sperm transfer.

Impacts on fertility, fecundity and offspring development

Supplementary Figure 4e presents these experimental protocols. To determine whether the decline in male reproductive fitness following heatwave exposure was a consequence of (1) reduced egg hatch (fertility), (2) reduced numbers of eggs produced (fecundity), or impacts on offspring development through the (3) larval and 4) pupal stages, we ran breeding assays to measure separate impacts on each (Supplementary Figure 4e). Males exposed to either 30 °C control or 42 °C heatwave conditions followed by 24 h at 30 °C were then paired monogamously with untreated and unmated females in 0.5 g flour and yeast topped with oats for 2 days at 30 °C. After mating, females were transferred to individual 4 ml vials with 0.5 g of pre-sieved flour and yeast topped with oats for oviposition under standard conditions. Every 2 days (and therefore before egg hatch) through a 10-day oviposition period, females were transferred to new vials, and eggs in the fodder sieved out using 300 μm mesh (Endecotts Ltd., London, UK). Separated eggs were dispersed on black tiles using a fine paintbrush and counted under a Zeiss Stemi 2000-C stereomicroscope at ×10 magnification to give a fecundity measurement for control (n = 59) and heatwave treatments (n = 76). For a random subset of the control (n = 40) and heatwave (n = 40) treatments, all eggs were returned immediately to 5 cm petri dishes containing 7 g of pre-sieved fodder to allow development. Ten days later, after which all successfully fertilised eggs would have hatched (egg development to hatch takes ~4 days under standard conditions in T. castaneum29, early stage larvae were sieved again from the fodder within each 2-day oviposition block and counted to provide egg hatch scores, before being returned to fodder. Twenty days later, pupae were counted in each block to quantify successful larval development and, at 35 days, when all hatched eggs, larvae and pupae would have developed to successful eclosion, adult offspring were counted.

Heatwave impacts on ejaculate sperm counts

Mature males were exposed to 5-day treatments of either 30 °C control (n = 36) or 42 °C heatwave (n = 56) conditions, then paired with a series of five mature untreated and unmated females in 1 cm2 mating arenas. Each male was paired with a female for 15 min, before being transferred to the next female. Access to a series of females allowed us to measure the rate of successful sperm transfer, and increased the probability that a male would transfer at least one spermatophore successfully to allow sperm counting (Supplementary Figure 4d). Immediately following each 15-min mating period, females were frozen at −20 °C for subsequent dissection and sperm count. Females were dissected in saline buffer (1% NaCl solution) under a Zeiss Discovery V.12 stereomicroscope (Carl Zeiss, Jena, Germany) under ×20 magnification. Using fine forceps, the female tract was removed, the bursa copulatrix cut open, and the tract then separated from any spermatophore which was isolated in 100 µl of saline buffer on a cavity slide. The spermatophore was then broken apart using size 0 dissection pins and the sperm mass released and dispersed into the buffer, before being washed off the slide and into a 10 ml tube using 3 ml of distilled water expelled from an autopipette. Each solution was then gently mixed before taking three 20 µl subsamples which were placed on flat glass slides to dry as smears. After air-drying, the slides were dipped gently into distilled water to remove any desiccant, and re-dried. Sperm cells (including their component parts, see below) adhere to the glass and were counted within each smear using dark field phase-contrast microscopy at ×200 magnification on an Olympus BX41 microscope (Olympus Corporation, Tokyo, Japan)72. Because many sperm cells had suffered membrane disruption and separation into their two elongate mitochondrial derivatives, possibly due to freeze damage, sperm number in each smear was determined by counting the total number of mitochondrial derivatives divided by two, added to the total number of undamaged sperm cells in each smear. The average sperm count for the three smears was then multiplied by their dilution factor (×155) to calculate total spermatophore sperm count.

Heatwave impacts on sperm migration in the female tract

Heatwave impacts on sperm function and distribution following insemination were assayed using males from a T. castaneum strain modified to incorporate a green fluorescent protein (GFP) into sperm chromatin43, enabling imaging of sperm distribution within the semi-transparent female reproductive tract (Fig. 3). Before mating, mature GFP males were exposed to 5-day treatments of either 30 °C control (n = 22) or 41 °C heatwave conditions (n = 24), followed by 24 h at 30 °C. Following treatment, GFP males were paired with mature untreated and standard KSS females for 90 mins. Following insemination, and to allow sperm to exit the spermatophore completely and reach longer-term storage in the bursa copulatrix and spermatheca42,43,64, females were snap-frozen 24 h after mating at -80 °C. The intact reproductive tracts of these females were then removed through microdissection of defrosted specimens under a Zeiss Discovery V.12 stereomicroscope (Carl Zeiss, Jena, Germany) in Grace’s insect buffer (Thermo Fisher, Massachusetts, USA). Following removal of the complete tract, the ovaries were separated from the upper tract, and the lower tract then excised from the oviduct’s junction with the ovipositor, keeping the main tract containing the bursa copulatrix, spermatheca and any sperm intact. This tract was then placed in 30 µl of Grace’s buffer on a slide and sealed under a 20 × 20 mm coverslip with impermeable instant contact adhesive (EVO-STIK, UK), before imaging using Zeiss Axiocam and Axiovision hardware and software.

Supplementary Figure 4d and 7 present these protocols. To visualise fluorescing sperm, brightfield and fluorescence images were acquired through a Zeiss ×10, 0.3 NA Plan-Neofluar objective on an AxioPlan 2ie microscope and captured with an Axiocam HRm CCD camera and Axiovision 4.8.2 software. Greater resolution of the smaller spermatheca was achieved through a Zeiss ×20, 0.6 NA Plan-Apochromat objective. GFP fluorescence, primarily from sperm, was excited through a 472 ± 15 nm excitation filter, and emitted fluorescence collected through a 520 ± 17.5 nm emission filter. General autofluorescence (AF) was excited through a 562 ± 20 nm excitation filter, and the emitted fluorescence collected using a 624 ± 20 nm filter. Exposure times were kept constant between samples. Images (14-bit greyscale) of the female tract and stored sperm were analysed using a custom-written macro in Fiji (ImageJ, ver. 1.49k)73 (Supplementary Figure 7). The macro subtracted background in each channel image using a rolling ball radius of 25 pixels for the smoothing algorithm74. To remove autofluorescence from the GFP-channel image so that only GFP sperm fluorescence was visible75, the macro corrected each GFP-channel image as follows: a region of interest (ROI) was created manually in the AF-channel in an area of the image displaying high fluorescence but no corresponding fluorescence in the GFP-channel image, the mean intensity was then measured in this ROI (Int Auto ). The typical structure for this ROI was the chitinous ring at the base of the spermathecal duct (Supplementary Figure 7c). The same ROI was then applied to the GFP-channel image and the mean intensity measured (Int GFP ). A correction factor (CF) was determined by dividing Int GFP by Int Auto . The AF-channel image was multiplied by CF and the resultant corrected AF image subtracted from the GFP-channel image, leaving only GFP sperm-derived fluorescence for measurement (Supplementary Figure 7d). The brightfield image was then used to define the ROI to be analysed by manually drawing around each tract’s perimeter walls (Supplementary Figure 7a, d). The mean pixel intensity within this ROI was then determined, providing a measure of the presence and distribution of GFP sperm in each tract.

Heatwave impacts on sperm viability

The impacts of heatwave conditions on mature sperm viability were measured from spermatophores transferred at mating to control females following exposures of mature males for 5 days at either 42 or 30 °C, and 24 h at 30 °C for both groups (Supplementary Figure 4d). Because males exposed to heatwaves can take longer to mate (Supplementary Figure 1), 42 °C heatwaved males were paired with untreated and unmated females for 210 min (n = 16) before dissection, and 30 °C control males for 90 mins (n = 10). Females were dissected immediately after their pairing period, with the protocol following that for sperm counts, apart from modifications for sperm viability staining and visualisation. Once spermatophores had been separated from the female bursa copulatrix, they were held in 30 µl of Grace’s insect buffer (Thermo Fisher, Massachusetts, USA) on a cavity slide. Having gently dispersed the sperm mass with size 0 dissection pins, sperm cells were stained with 2 µl of a 15-fold dilution of 2.4 mM propidium iodide and 2 µl of a 10-fold dilution 1 mM SYBER-14 dye from the LIVE/DEAD Sperm Viability Kit L-7011 (Molecular Probes, Oregon, USA). The sperm solutions were then sealed within the slide cavity using a 20 × 20 mm coverslip, and incubated for 5 mins at 27 ± 2 °C to allow stain uptake. Following incubation, image analysis took place using Zeiss Axiocam and Axiovision hardware and software. Sperm heads were imaged in (1) red and (2) green fluorescence channels, and (3) Differential Interference Contrast (for detecting non-stained sperm). All sperm observed in the viability assay took up the stain to fluoresce either red or green (Fig. 3).

Following staining and incubation using the LIVE/DEAD Sperm Viability Kit L-7011, differential-interference contrast and fluorescence images were acquired using a Zeiss ×20, 0.6 NA Plan-Apochromat objective on a AxioPlan 2ie microscope at ×200 magnification. Within 60 min of dissection, six images were captured at randomly selected locations across each diluted, incubated and stained sperm sample using a Axiocam HRm CCD camera. Propidium iodide fluorescence was excited using a 562 ± 20 nm excitation filter, and the emitted fluorescence collected with a 624 ± 20 nm filter. SYBER-14 fluorescence was excited with a 472 ± 15 nm excitation filter, and the emitted fluorescence collected through a 520 ± 17.5 nm emission filter. Using the L-7011 Sperm Viability Kit (Molecular Probes, Oregon, USA), live sperm with intact membranes take up the by SYBER-14 stain and their heads fluoresce green, while dead cells take up propidium iodide and fluoresce red. The proportion of viable sperm in each sample was calculated as the average (across the six subsamples) total number of live sperm, divided by the average total number of live sperm plus average total number of dead sperm. Counts were manual and based on colour dyed heads. Sperm survival has been previously shown to correlate with the number present76 therefore, sperm count was included as a random factor in a Generalised Linear Mixed Model77 (see Data Analysis).

Transgenerational impacts of heatwaves

Supplementary Figure 4f presents these experimental protocols. Consequences of heatwave conditions for the reproductive performance and lifespan of adult offspring in the next generation were measured following thermal exposure to males (sires), females (dams) and inseminated sperm held in female storage. Two assays were conducted to assess transgenerational heatwave effects on (1) offspring adult lifespan in both sexes, and (2) male offspring reproductive performance. Offspring mortality rates and lifespan were compared between adult offspring groups that had either been sired by males previously exposed to a 5-day heatwave at 40 °C (n = 28), or by control males exposed to 5 days at 30 °C (n = 29) (both groups held for 24 h at 30 °C before mating). Protocols to generate offspring followed those to measure reproductive fitness, after which adults were isolated individually in 4 ml vials with 0.5 g flour and yeast topped with oats under standard conditions at 30 °C. Mortalities were recorded and fodder refreshed every month for up to two years, after which all adult offspring had died. Lifespan was therefore measured in non-competitive and non-reproductive conditions, without adult interaction and with ad libitum food, providing a fair measure of intrinsic mortality in the absence of social, mating and environmental pressures. For each adult cross (40 °C heatwave n = 28 and 30 °C control n = 29), four adult offspring were randomly assigned and measured in the lifespan assay. Previous measures showed that sex ratios within offspring groups sired by males previous exposed to heatwave conditions did not depart from unity: average % male across n = 17 offspring groups = 51% (±2.36); Wilcoxon test of male proportion versus 0.5: V 17 = 79; P = 0.59.

In the second transgenerational fitness assay, we measured impacts of heatwaves in the previous generation on the reproductive performance of F 1 male offspring. Parental adults were either exposed to 42 °C heatwaves for 5 days followed by 24 h at 30 °C, or as 30 °C controls throughout. These control and heatwave treatments were exposed to both male and female adults to assess transgenerational effects upon male offspring reproductive fitness. Male (sire) effects were measured following exposure to 30 °C control (n = 42) and 42 °C (n = 48) heatwave conditions. Female (dam) and sperm-in-storage effects (dam + sperm) were measured following exposure to: (i) 30 °C control conditions in unmated females (dam alone control, n = 27), (ii) 42 °C heatwave conditions for unmated females (dam alone heatwave effect, n = 42), and (iii) 42 °C heatwave conditions for mated females carrying sperm in storage (dam plus sperm heatwave effect, n = 34). Following treatment, offspring were generated as in the reproductive output assays (Supplementary Figure 4a), and individual sons isolated at the pupal stage within those pairs producing offspring for subsequent assay. A single son was assayed from each of the parental crosses to standardise family effects. Because male T. castaneum have high reproductive potential65, we compared between treatment groups using an assay in which reproductive performance of individual males was measured following opportunities to mate with a series of 13 control unmated mature females, each provided to the male in 1 cm2 mating arenas for 30 min. After each 30-min access period, females were removed and exchanged for a new unmated female. Males were therefore tested for their ability to mate with and fertilise up to 13 females across a 6.5 h mating trial. Following each 30-min mating opportunity, females were transferred to 5 cm petri dishes for oviposition into 7 g flour and yeast, and 3 g of surface oats in standard 30 °C conditions across two 10-day blocks, as in the reproductive fitness assay (Supplementary Figure 4a). After oviposition, eggs were left to develop in standard conditions for 35 days, after which the total number of adult offspring produced, and the number of successful matings (evidenced by some offspring production), were counted. Our two scores of individual male reproductive performance were therefore: (1) the total number females successfully inseminated across the sequence of 13, and (2) the total number of offspring sired across the 6.5 h mating trial.

Data analysis

Data were analysed using R 3.3.278, using the RStudio.0.99.903 wrapper79. Graphs were produced using ‘ggplot{ggplot2}’80 package within R.

Descriptive statistics (mean ± S.E.) were calculated by ‘describeBy{psych}’81. Exploratory analysis included distribution plotting and conservative non-parametric testing on ranks prior to fitting generalised linear models (GLMs) with ‘glm{stats}82’. Heatwave treatments were entered into analyses as fixed factors. Where sampling structure variables (=blocks or experimental repeats) were present, either group averages were calculated, or generalised linear mixed models (GLMMs) were fitted83, using ‘glmer{lme4}’84. Cases where individuals died midway through assays were excluded.

The most appropriate error distribution for each GLM(M) was selected by examining diagnostic residual plots66,83,85 using ‘Plot{graphics}’86 and ‘mcp.fnc{LMERConvenienceFunctions}’87. Count response variables, which included all experiments measuring reproductive fitness, sperm counts, fecundity and number of mating events, were initially analysed using a Poisson distribution with a log link function. Model fits were checked and over-dispersion, where the variance exceeds the mean, was assessed in GLMs using by ‘dispersiontest{AER}’88, and in GLMMs using an over-dispersion function66. Where over-dispersion was present, usually due to zero-inflation in the heatwave treatments, corrections were applied by fitting a different error distribution (producing theta ~1)66. For moderate over-dispersion (1 < theta < 20), a quasi-Poisson error with a log link function was fitted. For strong over-dispersion (theta > 20), a negative binomial model with log link was fitted using ‘glm.nb{MASS}’89 (see Table 1 and Supplementary Table 1 for model errors and link functions). Continuous variables (mating duration and GFP sperm density distributions) were initially fitted using a Gaussian distribution with an identity link function, however, both had positively skewed residuals and outliers66. Model fits were improved for mating duration by using a log link function. Proportion response variables, which included paternity share in sperm competitions, sperm viability, and hatching, pupation and eclosion success, were fitted using a binomial distribution and a logit link function. Response variables were entered as a two column matrix of success-and-fail using cbind(success, fail){base}66. Where over-dispersion was present, usually due to zero-inflation in the heatwave treatments, it was corrected for by fitting a quasi-binomial distribution with a logit link function66. (See Table 1 and Supplementary Table 1 for model errors and link functions).

After each maximal model was fitted, the statistical significance of the experimental treatment variables were assessed using Akaike’s Information Criterion (AIC) comparisons, and log likelihood ratio tests (LLRT) with, and without, the term of interest83. The most efficient models had significantly lower AICs90,91. LLRTs were χ2 tests when the response variable was a count or proportion, and F tests when continuous66. LLRTs were primarily computed with ‘drop1{stats}’;66,78,83 ‘drop1{stats}’ was not compatible with quasi-error distributions, so was substituted for ‘lrtest{lmtest}’92. Simple post-hoc comparisons between treatment groups and controls were derived from summary(model)85,93. Post-hoc pairwise Tukey comparisons were applied using ‘lsmeans{lsmeans}’94. As a measure of how much variation in the response variable was explained by the model, pseudo R2 (explained deviance) was calculated for GLMs66. For GLMMs, ‘r.squaredGLMM{MuMIn}’95 reported the marginal R2 explained by the fixed factors, and conditional R2 for the fixed and random factors.