DOI: 10.5061/dryad.9s4mw6mtq
title: Data from: Does sexual dimorphism reflect sexual antagonism? Covariation of female fitness with brothers’ sexual traits and their female homologues in neriid flies

description: Alleles favoured by sexual selection in males can reduce fitness when
 expressed in females, resulting in intralocus sexual conflict. It remains
 unclear whether such conflict is fully resolved by the evolution of sexual
 dimorphism. If conflict persists, then female reproductive performance
 might covary negatively with the expression of secondary sexual traits in
 male relatives, and potentially with the expression of homologous traits
 in females themselves. However, because secondary sexual traits often
 exhibit strong condition dependence, a resource-poor developmental
 environment could weaken the covariance between female fitness and the
 expression of such traits. We tested these predictions using a split-brood
 experiment in which neriid flies (Telostylinus angusticollis) were reared
 on nutrient-rich or nutrient-poor larval diets, resulting in high (rich
 diet) and low (poor diet) adult sexual dimorphism. Consistent with
 predictions, in families reared on the rich larval diet, we found that
 females whose brothers exhibited relatively large secondary sexual traits
 produced fewer viable offspring. Moreover, rich-diet females with
 relatively large homologues of male sexual traits exhibited increased
 latency to oviposition. By contrast, in families reared on the poor larval
 diet, we found no evidence of negative covariation between male secondary
 sexual trait expression and female performance, and females with
 relatively large male trait homologues exhibited reduced latency to
 oviposition and increased fecundity. Our results confirm that sexually
 dimorphic morphology can reflect sexually antagonistic fitness variation,
 and suggest that intralocus sexual conflict remains unresolved in this
 species. Our results also suggest that the nutritional environment can
 modulate the signal of sexual antagonism.
description: <em>Experiment set-up</em>
 Approximately 30 neriid fly (<em>Telostylinus
 angusticollis</em>) individuals were collected from a natural
 population in Fred Hollows Reserve (33°54 44.04S 151°14 52.14E), Sydney,
 Australia, and mixed with stock flies (around 20 individuals) that had
 originated from the same location and had been reared in the lab for
 several generations. The flies were maintained in an 8L container with a
 mesh top and a layer of cocopeat on the bottom in a controlled-temperature
 room (~25 °C, 12 h light/dark cycle). The flies were supplied with food
 (sugar and yeast) ad libitum and watered periodically. To collect eggs, we
 provided oviposition medium in a large petri dish. The oviposition medium
 was prepared using rich larval food (described below) that was kept in a
 room at 25 °C for approximately seven days to allow mould growth, and
 watered and mixed periodically. From this lab population, 430 eggs were
 collected and raised on a standard larval diet in 500mL containers, with
 ~40 eggs per 200 g of food. The standard larval medium consisted of 10.9 g
 of soy protein (Nature’s Way brand, Pharm-a-care Pty. Ltd., Warriewood,
 NSW, Australia), 29.7 g of brown sugar (Coles brand, Bundaberg, Australia)
 and 500 mL of water per L of dry cocopeat. The larval medium was
 homogenized using a hand-held beater and frozen at –20°C until the day of
 use. The larval containers were incubated in a controlled-temperature
 chamber set to 25 °C ( 1.5 °C) and a 12 h light/dark cycle.
 After the F0 adult flies emerged, 100 male-female pairs were
 formed and placed in separate 200 mL vials with a substrate of moist
 cocopeat (Figure 4). After two weeks, when they reached maturity (Wylde et
 al. 2019), a small petri dish with oviposition medium was provided to each
 of the pairs, and the flies were given 5 days to oviposit. We collected
 eggs from pairs that laid at least 40 eggs (n = 62 pairs, 2,480 eggs in
 total). From each of these pairs, 20 eggs were transferred to a container
 with poor larval diet (20 eggs per 100 g of food) and 20 eggs were
 transferred to a container with a rich larval diet (20 eggs per 100 g of
 food). The poor diet consisted of 5.5 g of protein and 14.8 g of brown
 sugar, whereas the rich diet consisted of 32 g of protein and 89 g of
 brown sugar, both mixed with 500 mL of water and one L of dry cocopeat
 (Bonduriansky 2007a; Sentinella et al. 2013). The vials with the larval
 medium were placed inside one-L containers with a layer of cocopeat to
 facilitate pupation and collection of F1 adult flies (N = 1,148). The
 full-sibs descended from a male-female pair and split between rich and
 poor larval diets constitute a “family” in the analysis. A further 520
 eggs were collected from flies that were not used to produce the
 experimental families and reared on a standard larval diet to generate
 standard males for crosses used to assess female performance (see
 below). A maximum of 24 hours after emergence of the
 experimental flies (i.e., F1 flies from the 62 families) and standard
 flies from their puparia, males and females from each family were
 separated by sex to avoid mating. Upon emergence, two males randomly
 selected from each family were allowed 24 h for their exoskeletons to
 sclerotize fully and then frozen at −20°C for morphological measurements.
 We chose to measure two males from each family as there is little
 variation in body size or shape within families. Families that did not
 produce enough males or females were excluded from the analyses. Males
 were imaged using a Leica MC170HD camera mounted on a Leica MZ16A
 stereoscope (Wetzlar, Germany). For each male, we measured the lengths of
 the thorax as a proxy of body size, and the combined lengths of the head
 capsule and the right antenna as an index of secondary sexual trait
 expression, using ImageJ (Schneider et al. 2012). We used the residuals of
 the linear regression between family mean male secondary sexual trait
 length and family mean male thorax length to identify the F1 families with
 the lowest and highest expression of male secondary sexual traits relative
 to body size within each larval diet treatment group. Families with the
 largest negative residuals (12 families from rich larval diet and 10
 families from poor larval diet) or largest positive residuals (12 families
 from rich larval diet and 10 families from poor larval diet) were selected
 as focal families for experimental assays. From this
 subset of F1 families, two females (replicates) per family were randomly
 selected for the reproductive performance and longevity assays. Females
 were paired with standard males in separate 1 L containers and provided
 with oviposition medium. Females that developed in rich diet were paired
 with males at 10 +/- 2 days old, and females that developed in poor diet
 were paired with males at 20 +/- 2 days old as poor diet females take
 longer to reach reproductive maturity (Wylde et al. 2019). Eggs were
 counted every second day for eight days. From each female, twenty eggs
 (where possible) were transferred to containers with a standard diet (20
 eggs per 100 g of food) in order to quantify F2 offspring viability, a
 measure that incorporates egg hatching success and larval and pupal
 survival until the adult stage. We recorded the number of F2 adults that
 eclosed from the eggs over 14 days after the first adult eclosion in each
 replicate brood. To estimate F1 focal female longevity, the focal females
 were kept with their male partner in the same 1 L containers and provided
 with food (sugar and yeast) and water but not oviposition medium.
 Mortality was checked and recorded three times per week. Following death,
 the focal females were imaged and their head capsule length, antenna
 length and thorax length were measured from the images as described
 above. <em>Statistical
 analyses</em> We tested whether larval diet
 manipulation affected male and female body size using a Gaussian linear
 mixed model with thorax length as the dependent variable, larval diet as
 the predictor, and family identity as a random effect using the package
 glmmTMB (McGillycuddy et al. 2025). For this analysis, thorax length was
 standardized (z-transformed; mean = 0, standard deviation = 1) within
 sexes but across both larval diet treatments. We used a similar model to
 test larval diet effects on male and female body shape, quantified as head
 length/thorax length (using unstandardised morphological measurements). To
 test for covariation of relative head lengths of male and female siblings,
 we first carried out separate linear regressions of head length on thorax
 length (both variables standardised within sex × larval diet combinations)
 within each larval diet × sex combination and obtained the standardised
 residuals for each individual (representing individual relative head
 length). We then calculated the standardised family-mean residual for each
 sex within each larval diet. Finally, we fitted a Gaussian linear mixed
 model with family-mean female residual head length as the dependent
 variable and family-mean male residual head length, larval diet, and their
 interaction as fixed effects, as well as family identity as a random
 effect. We also fitted separate mixed models for each larval diet
 treatment group. We then tested for effects of
 brothers’ relative head length and focal female relative head length on
 focal female performance. We built linear mixed models that included
 larval diet, brothers’ relative head length (a categorical variable
 representing the sign of the family mean residual male head length),
 female relative head length (a continuous variable representing residual
 head length of each individual focal female), female body size (thorax
 length, standardized within larval diets to avoid redundancy with the
 categorical effect of larval diet), and the larval diet × brothers’
 residual head length and larval diet × female residual head length
 interactions, as fixed effects using glmmTMB. We used a Gaussian linear
 mixed model to test effects on the latency to lay eggs and generalized
 linear mixed models to test effects on the number of eggs laid (Poisson
 error distribution) and offspring viability (binomial error distribution,
 eggs that produced viable F2 adults versus eggs that did not produce
 viable F2 adults). We included an observation-level random effect to
 account for overdispersion when modelling the number of eggs laid. To test
 for effects on female survival, we fitted a Cox proportional hazards model
 using the package coxme (Therneau 2010). Family identity was included as a
 random effect in all models. Effects in glmmTMB and coxme models were
 tested using Wald z-tests. For dependent variables that yielded
 significant or near-significant interactions, we investigated the data
 further by carrying out post-hoc Tukey tests (for the larval diet ×
 brothers’ residual head length interaction) or separate analyses within
 each larval diet treatment group (for larval diet × female residual head
 length interactions). All statistical analyses were conducted using R
 version 4.4.2 (R_Core_Team 2021).
description: # Data from: Does sexual dimorphism reflect sexual antagonism? Covariation
 of female fitness with brothers’ sexual traits and their female homologues
 in neriid flies Dataset DOI:
 [10.5061/dryad.9s4mw6mtq](10.5061/dryad.9s4mw6mtq) ## Description of the
 data and file structure A split-brood larval diet manipulation experiment
 was carried out using the neriid fly Telostylinus angusticollis. Families
 from each larval diet treatment were ranked by the relative expression of
 male secondary sexual traits (head length relative to body size). From
 families with the largest and smallest male relative head length, focal
 females were randomly selected for fitness assays. The analysis tests for
 covariation between female fitness and the expression of females' own
 morphology (relative head length) and their brothers' morphology
 (relative head length).   ### Files and variables #### File: Script.R
 **Description:** R script to run the analyses #### File:
 female\_performance.csv **Description:**  ##### Variables *
 olre: observation-level random effect (unique code for each row) *
 diet: larval diet treatment (poor = nutrient-poor larval diet; rich =
 nutrient-rich larval diet) * family: F1 replicate family *
 totaleggs: total number of eggs laid by each F1 focal female over 8 days *
 latency: days from pairing with a male until the first egg was laid
 ("Latency to egg laying") * eggstransf: number of eggs
 transferred for assessment of offspring viability * emerged: number of
 viable F2 adult offspring obtained from the eggs transferred for
 assessment of offspring viability * notemerged: number of eggs that did
 not result in viable F2 adult offspring (calculated as eggstransf -
 emerged) * viability: proportion of eggs transferred that resulted in
 viable F2 adult offspring (emerged/eggstransf), used for plotting only *
 ageatdeath: F1 focal females' age at death (days) *
 femaleantennae: length of focal female antenna (arbitrary units) *
 femalehead: length of focal female head capsule (arbitrary units) *
 femaleheadtotal: sum of femaleantennae and femalehead (arbitrary units) *
 femalethorax: sum of femaleantennae and femalehead (arbitrary units) *
 femalethorax_std: femalethorax standardized (converted to z-scores) across
 both larval diets * femaleheadtotal_std: femaleheadtotal standardized
 (converted to z-scores) across both larval diets *
 femalethorax_stdbydiet: femalethorax standardized (converted to z-scores)
 within each larval diet treatment group *
 femaleheadtotal_stdbydiet: femaleheadtotal standardized (converted to
 z-scores) within each larval diet treatment group * femaleresid: mean
 standardised residual head length of each F1 focal female, calculated from
 a regression of total head length (head capsule length + antenna length)
 on thorax length; separate regressions were used within each larval diet
 treatment group * maleresidsign: categorical variable representing the
 sign of the family-mean standardised residual head length of two brothers
 from each focal family within each larval diet treatment; standaridised
 residual head length was calculated for each male from a regression of
 standardised total head length (head capsule length + antenna length) on
 standardised thorax length separately within each larval diet treatment
 group #### File: male\_measurements.csv **Description:**  ##### Variables
 * diet: larval diet treatment (poor = nutrient-poor larval diet; rich =
 nutrient-rich larval diet) * family: F1 replicate family * male_id: two
 brothers were measured from each replicate family; the numbers represent
 male identity * maleantennae: length of the male antenna (arbitrary units)
 * malehead: length of the male head capsule (arbitrary units) *
 malethorax: length of the male thorax (arbitrary units) *
 maleheadtotal: sum of maleantennae and malehead (arbitrary units) *
 malethorax_std: malethorax standardized (converted to z-scores) across
 both larval diets * maleheadtotal_std: maleheadtotal standardized
 (converted to z-scores) across both larval diets *
 malethorax_stdbydiet: malethorax standardized (converted to z-scores)
 within each larval diet treatment group *
 maleheadtotal_stdbydiet: maleheadtotal standardized (converted to
 z-scores) within each larval diet treatment group #### File:
 mean\_residuals\_by\_family.csv **Description:**  ##### Variables *
 diet: larval diet treatment (poor = nutrient-poor larval diet; rich =
 nutrient-rich larval diet) * family: F1 replicate family *
 femaleresidual: mean standardised residual head length of the two focal
 females (sisters) from each family x larval diet treatment combination;
 standaridised residual head length was calculated for each focal female
 from a regression of total head length (head capsule length + antenna
 length) on thorax length; separate regressions were used within each
 larval diet treatment group * maleresidual: mean standardised residual
 head length of two brothers from each family x larval diet treatment
 combination; standaridised residual head length was calculated for each
 male from a regression of total head length (head capsule length + antenna
 length) on thorax length; separate regressions were used within each
 larval diet treatment group ## Code/software R version 4.4.2