Dataset DOI: 10.5061/dryad.1g1jwsvbk

Description of the data and file structure

Title of the dataset:"Parental social environment has no effect on offspring development in the dung beetle: A test of adult sex ratio effects"

Dataset description
We have deposited the following raw data files and analysis code in this dataset:

Files and variables

File: brood_balls_weight.csv

Description: For this dataset, we quantified parental investment allocated to offspring as the actual dry mass of brood balls. Brood balls originating from different ASR treatments (female-biased, unbiased, and male-biased) were carefully cleaned of adhering soil and fecal impurities, oven-dried to a constant mass at 60 °C for 48 hours, and then precisely weighed. We compared the magnitude of parental investment across ASR treatments to test whether parents adjust their investment strategies in response to social stress imposed by skewed ASR conditions.

Variables

• obs: Generic data ID for data management and sorting only; no substantive experimental or biological value, not used in statistical analyses.
• treatment: Categorical variable for ASR treatment, with 3 levels: female-biased (FB), unbiased (UB), male-biased (MB).
• group: Unique ID for each independent experimental replicate.
• id: Unique ID for each individual brood ball, corresponding to the offspring used in subsequent developmental assays.
• brood_balls_weight: Dry mass of each individual brood ball

File: contest_and_courtship_frequency.csv

Description: To verify that social environments with distinct adult sex ratios (ASRs) exert measurable effects on individual contest behaviours and male courtship behaviours within the population, we established three experimental ASR treatments: female-biased, unbiased, and male-biased. We focused our comparisons on the frequencies of contest and courtship events recorded per observation arena between the female-biased and male-biased conditions. These data were used to quantify the intensity of parental contest and courtship behaviours across different ASR regimes, and to confirm that population ASR is a key determinant shaping social and sexual interaction dynamics in this species.

Variables

• treatment: Categorical variable for ASR treatment, with 3 levels: female-biased (FB), unbiased (UB), male-biased (MB).
• group: Unique ID for each independent experimental replicate.
• behavior_type: Categorical variable for behavioural event type: contest behaviour courtship behaviour.
• times: Total count of behavioural events

File: MANOVA.csv

Description: To examine whether parental exposure to ASR-mediated social stress induces significant alterations in the duration of distinct offspring developmental stages, we tracked the developmental progression of larvae from all three ASR treatment groups under standardized common-garden rearing conditions. For each individual offspring, we quantified the duration of the larval stage, pupal stage, and total developmental period from hatching to adult emergence. We then fitted a multivariate analysis of variance (MANOVA) model to these correlated developmental traits to assess the effects of parental ASR experience on offspring developmental timing.

Variables

• obs: Generic data ID for data management and sorting only; no substantive experimental or biological value, not used in statistical analyses.
• treatment: Categorical variable for ASR treatment, with 3 levels: female-biased (FB), unbiased (UB), male-biased (MB).
• group: Unique ID for each independent experimental replicate.
• id: Unique ID for each individual offspring.
• sex: Categorical variable for offspring sex: male / female.
• stage_larvae: Duration of the larval stage, from hatching to pupation.
• stage_pupa: Duration of the pupal stage, from pupation to adult emergence.
• emerging_time: Total developmental duration from hatching to adult emergence, sum of larval and pupal stages.

File: development_speed_and_emerging_weight.csv

Description: To investigate whether parental exposure to ASR-mediated social stress modulates offspring metamorphic development, we longitudinally tracked the developmental trajectories of offspring derived from each of the three ASR treatment groups. First, we collected all brood balls produced by breeding populations under the different ASR regimes. We then individually transferred fertilized eggs from their original brood balls into standardized artificial brood balls, which provided highly uniform microenvironmental and nutritional conditions for all individuals across treatments. This common-garden rearing design eliminates confounding effects of variable parental provisioning, allowing us to isolate potential germline-mediated transgenerational effects of parental ASR experience on offspring development. We non-invasively opened the artificial brood balls at 48-hour intervals post-hatching to weigh each larva and record its developmental stage. This monitoring was repeated for every individual throughout the entire developmental period until adult emergence, when we also documented the sex of each newly emerged beetle.

Variables

• obs.: Sequential observation ID for data sorting only; no substantive experimental value, not used in statistical analyses.
• treatment: Categorical variable for ASR treatment, with 3 levels: female-biased (FB), unbiased (UB), male-biased (MB).
• group: Unique ID for each independent experimental replicate.
• id: Unique ID for each individual offspring.
• sex: Categorical variable for offspring sex: male / female.
• summit_weight: Maximum body mass attained by each offspring during development.
• days_reaching_summit: Number of days from hatching to attaining peak body mass.
• weight_of_emerging_beetle: Body mass of each offspring measured immediately after adult emergence.
• body_size_of_emerging_beetles: Body size of newly emerged adult offspring.
• horn_size_of_emerging_beetle: Horn size of newly emerged adult offspring.
• stage_larvae: Duration of the larval stage, from hatching to pupation.
• stage_pupa: Duration of the pupal stage, from pupation to adult emergence.
• emerging_time:Total developmental duration from hatching to adult emergence, sum of larval and pupal stages.
• speed: Offspring developmental growth rate, calculated as peak body mass divided by days to peak mass.

File: development_speed_and_emerging_weight_drawing.csv

Description: This file serves solely as supporting material for figure generation and is not intended for formal statistical analyses. It contains the same core dataset as the main developmental file, formatted for streamlined data visualization.

Variables

• obs: Sequential observation ID for data visualization and sorting only; no substantive analytical value.
• treatment: Categorical variable for ASR treatment, with 3 levels: female-biased (FB), unbiased (UB), male-biased (MB).
• group: Unique ID for each independent experimental replicate.
• id: Unique ID for each individual offspring.
• Sex: Categorical variable for offspring sex: male / female.
• days: Measurement day, calculated as days post larval hatching.
• stage:Categorical variable for offspring developmental stage, with 3 levels: larval stage, pupal stage, emerging stage.
• weight: Fresh body mass of the individual at each measurement point.

File: R_code.r

Description: This file contains the complete R code used to conduct all statistical analyses reported in the associated research article. All code was developed and validated in R version 4.3.1, consistent with the analytical methods described in the manuscript.

The script includes code for loading and cleaning raw datasets, fitting linear mixed-effects models (LMMs), conducting post-hoc multiple comparisons, and producing all result figures. All analyses were implemented in R v4.3.1, with dependencies including lme4, lmerTest, DHARMa, emmeans, and multcomp.

For detailed documentation of the analytical procedures, model specifications, and statistical frameworks, please refer to the Data analysis section of our paper.

Code/software

R is required to run "R code.r"; the script was created using version 4.3.1 (R Core Team, 2023). Line-level annotations are added across the entire script, organized to correspond to the four major components of the analytical workflow: 1) loading dependent packages, 2) data loading and preprocessing, 3) statistical analysis procedures, and 4) figure plotting and output.

Statistical Analyses Overview
All analyses were conducted in R v4.3.1 using linear mixed-effects models (LMMs) fitted via the lme4 package, with fixed-effect P-values calculated by lmerTest and model assumptions (residual normality, homoscedasticity) verified via DHARMa simulation diagnostics. Model selection followed the Akaike Information Criterion (AIC): non-significant interaction terms were removed for simplification while all main effects were retained. Experimental replicate (group) and/or individual offspring ID were included as random intercepts to account for the hierarchical data structure; post hoc pairwise comparisons were run with emmeans and adjusted for multiple testing via multcomp.

Behavioural frequency analysis
File: contest_and_courtship_frequency.csv
LMMs tested effects of ASR treatment, behaviour type, and their interaction on behavioural event counts, with experimental group as a random intercept.

Brood ball mass analysis
File: brood_balls_weight.csv
An LMM compared dry brood ball mass across ASR treatments, with experimental group as a random intercept.

Offspring developmental analysis
Files: development_speed_and_emerging_weight.csv, MANOVA.csv
Growth & emergence traits: Separate LMMs assessed effects of ASR treatment and sex on developmental speed and emergence body mass, with experimental group as a random effect.

Stage-specific duration: Long-format LMM included ASR treatment, sex, developmental stage, and their interactions as fixed effects, with group and individual ID as random effects to control for repeated measures.