Data for: Does sexual dimorphism reflect sexual antagonism? Covariation of female fitness with brothers' sexual traits and their female homologues in neriid flies

Proceedings of the Royal Society of London B Does sexual dimorphism reflect sexual antagonism? Covariation of female fitness with brothers’ sexual traits and their female homologues in neriid flies Vasconcelos, A.C.O. & Bonduriansky, R. Abstract 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 less 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. MATERIAL AND METHODS Experiment set-up Approximately 30 neriid fly (Telostylinus angusticollis) 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 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. Statistical analyses 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).《伦敦皇家学会会报B辑》（"Proceedings of the Royal Society of London B"） 性别二态性是否反映性拮抗？狭颈狭颜蝇（Telostylinus angusticollis）雌适合度与其雄性亲属的性征及其雌性同源性状的协变关系 瓦斯康塞洛斯 A.C.O. 与 邦杜里安斯基 R. 摘要 性选择青睐的等位基因在雌性中表达时会降低适合度（fitness），从而引发基因座内性冲突（intralocus sexual conflict）。目前尚不明确这类冲突是否可通过性别二态性（sexual dimorphism）的演化得到完全解决。若冲突持续存在，雌性繁殖性能可能与雄性亲属的第二性征（secondary sexual traits）表达呈负相关，也可能与雌性自身同源性状的表达相关联。然而，由于第二性征通常表现出强烈的条件依赖性（condition dependence），资源匮乏的发育环境可能会削弱雌性适合度与这类性状表达之间的协变关系。我们通过分群实验（split-brood experiment）检验了上述预测：以狭颈狭颜蝇为研究对象，将其幼虫饲养在营养丰富或营养匮乏的日粮中，分别获得成虫期高（丰裕日粮组）和低（匮乏日粮组）的性别二态性。结果与预测一致：在丰裕幼虫日粮组的家系中，雄性兄弟第二性征相对较大的雌性，其后代存活力更低；此外，丰裕日粮组中雄性性征同源性状相对较大的雌性，产卵潜伏期延长。与之形成对比的是，在匮乏幼虫日粮组的家系中，未发现雄性第二性征表达与雌性繁殖性能存在负协变关系的证据，且雄性性征同源性状相对较大的雌性，其产卵潜伏期缩短、繁殖力提升。我们的研究结果证实，性别二态形态可反映性拮抗（sexual antagonism）适合度变异，并表明该物种的基因座内性冲突仍未得到解决。此外，本研究还提示，营养环境可调控性拮抗的信号强度。 材料与方法 实验设计 我们从澳大利亚悉尼弗雷德·霍洛克斯保护区（33°54′44.04"S，151°14′52.14"E）的自然种群中采集了约30只狭颈狭颜蝇个体，并与源自同一地点、在实验室饲养多代的原种蝇（约20只）混合饲养。将果蝇饲养于容积8L的容器中，容器顶部带有网纱，底部铺有椰糠，饲养环境为控温房间（约25℃，12 h光暗循环）。自由提供糖和酵母作为食物，并定期补水。为收集卵粒，我们在大型培养皿中放置产卵介质：产卵介质采用下述丰裕幼虫日粮制备，置于25℃环境中约7天以促进霉菌生长，并定期补水混匀。从该实验室种群中采集430枚卵，以每200g食物对应约40枚卵的密度，在500mL容器中使用标准幼虫日粮饲养。标准幼虫培养基每升干椰糠对应：大豆蛋白（Nature’s Way品牌，Pharm-a-care Pty. Ltd.，沃里伍德，新南威尔士州，澳大利亚）10.9g、红糖（Coles品牌，班达伯格，澳大利亚）29.7g、水500mL。使用手持搅拌器将幼虫培养基均质化，于-20℃冷冻保存至使用当日。幼虫饲养容器置于控温培养箱中，设置为25℃（±1.5℃）、12 h光暗循环。 F0代成虫羽化后，组建100对雌雄配对，分别置于200mL的小瓶中，瓶内铺有湿润椰糠作为基质（图4）。两周后，当果蝇达到性成熟（Wylde等，2019），向每对果蝇提供装有产卵介质的小型培养皿，给予5天时间产卵。收集至少产卵40枚的配对（共62对，总计2480枚卵）。从每对配对中各取20枚卵，分别转移至添加匮乏幼虫日粮的容器（每100g食物对应20枚卵）和丰裕幼虫日粮的容器（每100g食物对应20枚卵）。匮乏日粮组成分为：蛋白质5.5g、红糖14.8g；丰裕日粮组成分为：蛋白质32g、红糖89g，两组均混合500mL水与1L干椰糠（Bonduriansky 2007a；Sentinella等，2013）。将装有幼虫培养基的小瓶置于容积1L的容器中，容器底部铺有椰糠以方便化蛹和收集F1代成虫（共1148只）。源自同一雌雄配对且被分配至丰裕、匮乏幼虫日粮的全同胞家系（full-sibs），即为本分析中的“家系”。另外从未用于组建实验家系的果蝇中采集520枚卵，以标准幼虫日粮饲养，用于产生用于交配实验的标准雄蝇，以评估雌性繁殖性能（详见下文）。 实验蝇与标准蝇处理 实验蝇（即来自62个家系的F1代）与标准蝇从蛹壳中羽化后的24小时内，按性别分离各家庭的雌雄个体以避免交配。羽化后，从每个家系中随机选取2只雄蝇，使其外骨骼充分骨化24小时，随后置于-20℃冷冻以备形态学测量。选择每家庭2只雄蝇的原因是家系内个体的体型或形状变异极小。无法提供足够雄性或雌性个体的家系将被排除在分析之外。使用安装在Leica MZ16A体视显微镜（德国韦茨拉尔）上的Leica MC170HD相机对雄蝇成像。使用ImageJ（Schneider等，2012）软件，测量每只雄蝇的胸部长度作为体型代理指标，并以头壳与右侧触角的总长度作为第二性征表达的指数。我们以家系平均雄性第二性征长度与家系平均雄性胸部长度的线性回归残差，来识别每个幼虫日粮处理组中，相对于体型而言第二性征表达最低和最高的F1家系。选取残差最大负值的家系（丰裕日粮组12个，匮乏日粮组10个）与残差最大正值的家系（丰裕日粮组12个，匮乏日粮组10个），作为实验测定的焦点家系。 从上述焦点F1家系子集内，每个家系随机选取2只雌性个体（生物学重复）用于繁殖性能与寿命测定。将雌性个体与标准雄蝇配对，置于独立的1L容器中，并提供产卵介质。丰裕日粮组发育的雌性在10±2日龄时与雄蝇配对，匮乏日粮组发育的雌性在20±2日龄时配对，因为匮乏日粮组雌性达到繁殖成熟的时间更长（Wylde等，2019）。每两天统计一次卵粒数量，持续8天。从每只雌性个体中，尽可能转移20枚卵至标准日粮容器（每100g食物对应20枚卵），以量化F2代后代存活力，该指标涵盖卵孵化成功率、幼虫与蛹的存活率直至成虫阶段。记录每个重复家系中，从首次成虫羽化起14天内孵化出的F2代成虫数量。为估算F1代焦点雌性的寿命，将焦点雌性与其雄性伴侣共同饲养在同一1L容器中，自由提供糖、酵母与水，但不提供产卵介质。每周三次检查并记录死亡情况。雌性个体死亡后，对其进行成像，并按照前述方法测量头壳长度、触角长度与胸部长度。 统计分析 我们使用高斯线性混合模型（Gaussian linear mixed model）检验幼虫日粮操控对雌雄体型的影响，以胸部长度为因变量，幼虫日粮为预测因子，家系身份为随机效应，分析软件使用glmmTMB（McGillycuddy等，2025）。本分析中，胸部长度在雌雄个体间、两个幼虫日粮处理组内进行标准化（z变换；均值=0，标准差=1）。我们使用类似模型检验幼虫日粮对雌雄体型形状的影响，体型形状以头长/胸长量化（使用未标准化的形态学测量值）。为检验雄性与雌性同胞的相对头长协变关系，我们首先在每个幼虫日粮×性别组合内，分别进行头长对胸长的线性回归（两个变量均在性别×幼虫日粮组合内标准化），并获得每个个体的标准化残差（代表个体相对头长）。随后，我们计算每个幼虫日粮组内，每个性别的家系平均残差的标准化值。最后，我们拟合高斯线性混合模型，以家系平均雌性残差头长为因变量，家系平均雄性残差头长、幼虫日粮及其交互项为固定效应，家系身份为随机效应。我们还针对每个幼虫日粮处理组分别拟合了混合模型。 随后，我们检验了雄性兄弟相对头长与焦点雌性相对头长对焦点雌性繁殖性能的影响。我们构建了线性混合模型，固定效应包括：幼虫日粮、雄性兄弟相对头长（代表家系平均雄性残差头长符号的分类变量）、雌性相对头长（代表每只焦点雌性个体残差头长的连续变量）、雌性体型（胸部长度，在幼虫日粮组内标准化以避免与幼虫日粮分类效应产生冗余）、幼虫日粮×雄性兄弟残差头长以及幼虫日粮×雌性残差头长的交互项，分析软件使用glmmTMB。我们使用高斯线性混合模型检验对产卵潜伏期的影响，使用广义线性混合模型（generalized linear mixed models）检验对产卵数量（泊松误差分布）与后代存活力（二项误差分布，即产生可育F2代成虫的卵与未产生可育F2代成虫的卵）的影响。在建模产卵数量时，我们纳入了观测水平随机效应以处理过度离散问题。为检验对雌性存活的影响，我们使用coxme软件包拟合Cox比例风险模型（Cox proportional hazards model，Therneau，2010）。所有模型中均纳入家系身份作为随机效应。使用Wald z检验对glmmTMB与coxme模型中的效应进行显著性检验。对于存在显著或接近显著交互作用的因变量，我们进一步开展后续分析：针对幼虫日粮×雄性兄弟残差头长的交互项进行事后Tukey检验，针对幼虫日粮×雌性残差头长的交互项分别在每个幼虫日粮处理组内进行分析。所有统计分析均使用R版本4.4.2（R_Core_Team，2021）完成。