Avian predators avoid attacking fly-mimicking beetles: A field experiment on evasive mimicry using artificial prey - Dataset & R Scripts

Avian predators avoid attacking fly-mimicking beetles: A field experiment on evasive mimicry using artificial prey

Many Neotropical beetles present coloration patterns mimicking red-eyed flies, which are presumably evasive mimicry models. However, the role of predators in selecting for evasive mimics in nature remains untested. In a field experiment, we used nontoxic plasticine replicas of a specialized fly-mimicking beetle species, which we placed on the host plants of the beetles. We show that replicas painted with reddish patches simulating the eyes of flesh flies experienced a much lower predation rate than control replicas. We found that beak marks were the most frequent signs of attack on plasticine replicas, underlining the potential selective pressure exerted by birds. Replicas that matched the size of the beetles suffered higher predation than smaller or larger replicas. The predation rate was also higher for beetle replicas exposed during the warm and wet season, when adult beetles occur. Our results support predator-mediated selection of mimic beetles, highlighting that reddish spots resembling flies’ eyes comprise an important trait in reducing attack by avian predators.
This study describes the results of two field experiments using artificial beetle replicas made with nontoxic plasticine placed on host plants of a specialized fly-mimicking beetle species.
The experiment_#1 manipulated the coloration pattern of beetle replicas to test the prediction that replicas with red patches would suffer less attacks by avian predators than control replicas not resembling red-eyed flies.
The experiment_#2 evaluated three specific predictions.
First, attack marks corresponding to beaks should be frequently observed in the beetle replicas.
Second, we predicted that replicas with intermediate size (0,5 – 1,0 cm) should be more attacked by birds than those in the extreme size classes (0,25 – 2,0 and 4,0 cm).
Third, we predicted that avian attacks on replicas should be higher in the peak of the wet season than during the peak of the dry season.

The file "Dataset_Avian predators avoid attacking fly-mimicking" contains all data.
The spreadsheet Experiment_#1_raw contains seven columns and 361 lines, where each line represents a single plasticine replica.
The first column (#), designates plasticine replica number within each sample unity.
The second column (Site) designates the area where blocks were stablished.
The third column (Block) designates different locations where sample unities
(groups of 10 plasticine replicas of each treatment) were subject to same spatial conditions.
The fourth column (PlantTag) designates individual mistletoes where replicas were placed.
The fifth column (Treatment) designates coloration pattern of plasticine replicas, classified in control, bad (brown-eyed) and good (red-eyed).
The sixth column (Attack) designates the occurrence of attack to replicas, and classification according to vestiges left on plasticine (bird - V or U-shaped beak marks; insects - small mouthparts perforations; mammal - teeth marks; missing - plasticine removed).

The spreadsheet Experiment_#1 contains five columns and 37 lines, where each line represents a sample unity,
group of ten plasticine replicas of the same color pattern (Treatment) within a block.
The first column (Site) designates the area where blocks were stablished.
The second column (Block) designates different locations of groups of 10 plasticine replicas of each treatment.
The third column (Treatment) designates color pattern of plasticine replicas.
The fourth column (Total) designates the valid number of replicas in each sample unity.
The fifth column (Attacked) designates the number of replicas with evidence of bird attack.

The spreadsheet Experiment_#2_raw contains eight columns and 1201 lines, where each line represents a single plasticine replica.
The first column (#), designates plasticine replica number within each sample unity.
The second column (Site) designates the area where blocks were stablished.
The third column (Block) designates different locations where sample unities
(groups of 10 plasticine replicas of each treatment) were subject to same spatial conditions.
The fourth column (PlantTag) designates individual threes where replicas were placed.
The fifth column (Season) designates period when the experiment was conducted.
The sixth column (Size) designates size class of plasticine replicas.
The seventh column (Attack) designates the occurrence of attack to replicas, and classification according to vestiges left on plasticine (bird - V or U-shaped beak marks; insects - small mouthparts perforations; mammal - teeth marks; missing - plasticine removed).

The spreadsheet Experiment_#2 contains seven columns and 121 lines, where each line represents a sample unity, group of ten plasticine replicas of the same size within a block in each period.
The first column (Site) designates the area where blocks were stablished.
The second column (Block) designates different locations of groups of 10 plasticine replicas of each treatment.
The third column (Season) designates period when the experiment was conducted.
The fourth column (Size) designates size class of plasticine replicas.
The fifth column (Total) designates the valid number of replicas in each sample unity.
The sixth column (Attacked) designates the number of replicas with evidence of bird attack.

The spreadsheet Coordinates contains blocks’ geographical coordinates and elevation data.
We analyzed data using R software (R Core Team, 2019).
We employed generalized linear mixed effects models (GLMMs, glmer for non-normal datasets, with lme4 package in R) with fixed and random effects to analyze the datasets of replica attacks for experiments #1 and #2.

In the experiment_#1, the explanatory variable Treatment encompassed replica coloration with three levels
(control – nor paint, bad – brown eyed, and good – red eyed) and was considered as a fixed factor and Site and Block assigned as random effect.
The response variable were the proportions of replicas exclusively attacked by avian predators after 14 days of exposure, calculated by dividing the variable Attacked by the variable Total.
For this analysis we used data of the spreadsheet Experiment_#1.
Data of spreadsheet Experiment_#1 was transformed into a TXT file denominated "Attack" to be used in R software.
In the experiment_#2 the explanatory variables Size (replica size classes, five levels: 0,25 – 0,5 – 1,0 – 2,0 – 4,0 cm) and Season (two levels: dry and wet) and the interaction among these factors were considered as fixed factors, whereas Site and Block were assigned as random effects accounting for spatial heterogeneity of samples.
The response variable were the proportions of replicas exclusively attacked by avian predators after 14 days of exposure, calculated by dividing the variable Attacked by the variable Total.
For this analysis we used data of the spreadsheet Experiment_#2.
Data of spreadsheet Experiment_#2 was transformed into a txt.file denominated "Size" to be used in R software.
The dataset of both experiments fitted binomial distribution error of the response variable.
We selected the minimal models after the removal of non-significant variables (P-value > 0.05).
If we detected significant differences in variables with more than two levels (Size or Treatment),
we performed contrast analysis to determine differences among levels.
In both minimal models we checked for error distribution and data overdispersion.
The figures 3 and 4ab were generated on Excel using absolute counts or claculated means and standard errors.
The raw data underlying Figures 3, 4a and 4b are on the spreadsheets with the same name (Figure 3, Figure 4a, Figure 4b) of file "Dataset_Avian predators avoid attacking fly-mimicking", where the figure and the underlying formula are located. Double click on the figures to view how the data generated the figure.
The figures were pasted on PowerPoint for inclusion of letters and photos of the replicas.
Slides were than transformed on .jpeg archives.

鸟类捕食者规避攻击拟蝇甲虫：一项基于人工猎物的躲避拟态野外实验

许多新热带区（Neotropical）甲虫演化出模仿红眼蝇的体色模式，这类模式被认为属于躲避拟态（evasive mimicry）的典型范例。然而，自然界中捕食者对躲避拟态甲虫的选择作用尚未得到实证检验。

本研究开展野外实验，以一种专性拟蝇甲虫的无毒橡皮泥（plasticine）复制品为实验材料，将其放置于该甲虫的寄主植物上。实验结果显示，模拟肉蝇（flesh fly）眼部的红色斑块涂装复制品的被捕食率显著低于对照组复制品。我们观察到，橡皮泥复制品上最常见的攻击痕迹为鸟类喙部留下的印痕，这印证了鸟类施加的潜在选择压力。与甲虫实际体型匹配的复制品的被捕食率高于体型过小或过大的复制品。在成虫出现的温暖湿季放置的甲虫复制品，其被捕食率同样更高。本研究结果支持捕食者介导的拟态甲虫选择作用，同时表明模拟蝇类眼部的红色斑点是降低鸟类捕食者攻击的关键性状。

本研究详述了两项野外实验的结果：实验以专性拟蝇甲虫的无毒橡皮泥复制品为材料，将其放置于该甲虫的寄主植物上开展实验。

实验1（experiment_#1）通过调控甲虫复制品的体色模式，验证如下假说：带有红色斑块的复制品的鸟类捕食者攻击率，将低于未模拟红眼蝇的对照组复制品。

实验2（experiment_#2）则验证三项具体假说：其一，甲虫复制品上应频繁出现鸟类喙部留下的攻击痕迹；其二，体型处于中间区间（0.5–1.0厘米）的复制品的鸟类攻击率，将高于极端体型区间（0.25–2.0厘米与4.0厘米）的复制品；其三，湿季峰值时段放置的复制品的鸟类攻击率，将高于干季峰值时段的复制品。

所有实验数据均存储于名为"Dataset_Avian predators avoid attacking fly-mimicking"的数据集文件中。

原始数据表Experiment_#1_raw（实验1原始数据表）共包含7列、361行，每一行对应单个橡皮泥复制品。第1列（编号，#）标注每个样本单元内橡皮泥复制品的编号；第2列（采样点，Site）标注区块设置的区域；第3列（区块，Block）标注不同的实验点位，每个点位内的样本单元（每组包含10个对应处理组的橡皮泥复制品）处于相同的空间条件下；第4列（植物标签，PlantTag）标注复制品所附着的单株槲寄生；第5列（处理组，Treatment）标注橡皮泥复制品的体色模式，分为对照组（未涂装）、劣组（棕眼）与优组（红眼）；第6列（攻击情况，Attack）标注复制品是否遭受攻击，并根据橡皮泥上的痕迹进行分类：鸟类攻击（V形或U形喙痕）、昆虫攻击（小型口器穿孔）、哺乳动物攻击（齿痕）、缺失（橡皮泥被完全移除）。

汇总数据表Experiment_#1（实验1汇总表）共包含5列、37行，每一行对应一个样本单元——即单个区块内，10个具有相同体色模式（处理组）的橡皮泥复制品组成的组。第1列（采样点，Site）标注区块设置的区域；第2列（区块，Block）标注每个处理组10个橡皮泥复制品所在的不同点位；第3列（处理组，Treatment）标注橡皮泥复制品的体色模式；第4列（有效总数，Total）标注该样本单元内的有效复制品数量；第5列（受攻击数量，Attacked）标注该样本单元内存在鸟类攻击痕迹的复制品数量。

原始数据表Experiment_#2_raw（实验2原始数据表）共包含8列、1201行，每一行对应单个橡皮泥复制品。第1列（编号，#）标注每个样本单元内橡皮泥复制品的编号；第2列（采样点，Site）标注区块设置的区域；第3列（区块，Block）标注不同的实验点位，每个点位内的样本单元（每组包含10个对应处理组的橡皮泥复制品）处于相同的空间条件下；第4列（植物标签，PlantTag）标注复制品所附着的单株树木；第5列（季节，Season）标注实验开展的时段；第6列（体型，Size）标注橡皮泥复制品的体型区间；第7列（攻击情况，Attack）标注复制品是否遭受攻击，并根据橡皮泥上的痕迹进行分类：鸟类攻击（V形或U形喙痕）、昆虫攻击（小型口器穿孔）、哺乳动物攻击（齿痕）、缺失（橡皮泥被完全移除）。

汇总数据表Experiment_#2（实验2汇总表）共包含7列、121行，每一行对应一个样本单元——即单个时段、单个区块内，10个具有相同体型的橡皮泥复制品组成的组。第1列（采样点，Site）标注区块设置的区域；第2列（区块，Block）标注每个处理组10个橡皮泥复制品所在的不同点位；第3列（季节，Season）标注实验开展的时段；第4列（体型，Size）标注橡皮泥复制品的体型区间；第5列（有效总数，Total）标注该样本单元内的有效复制品数量；第6列（受攻击数量，Attacked）标注该样本单元内存在鸟类攻击痕迹的复制品数量。

坐标数据表Coordinates（坐标表）包含各实验区块的地理坐标与海拔数据。

本研究使用R软件（R software, R Core Team, 2019）进行数据分析：针对实验1与实验2的复制品攻击数据，采用包含固定效应与随机效应的广义线性混合模型（generalized linear mixed effects models, GLMMs，使用R语言的lme4包中的glmer函数处理非正态数据集）进行分析。

在实验1中，解释变量为处理组（Treatment），该变量对应复制品的体色模式，包含3个水平：对照组（未涂装）、劣组（棕眼）与优组（红眼），被设定为固定效应；采样点（Site）与区块（Block）则被设定为随机效应，以控制空间异质性。本研究的响应变量为暴露14天后仅遭受鸟类捕食者攻击的复制品比例，计算方式为‘受攻击数量（Attacked）’除以‘有效总数（Total）’。本次分析使用实验1汇总表的数据，并将该数据表转换为名为"Attack"的文本文件以供R软件调用。

在实验2中，解释变量包括体型（Size，复制品体型区间共5个水平：0.25–0.5–1.0–2.0–4.0厘米）、季节（Season，包含2个水平：干季与湿季），以及二者的交互效应，上述变量均被设定为固定效应；采样点与区块则被设定为随机效应，以控制样本的空间异质性。本研究的响应变量同样为暴露14天后仅遭受鸟类捕食者攻击的复制品比例，计算方式为‘受攻击数量’除以‘有效总数’。本次分析使用实验2汇总表的数据，并将该数据表转换为名为"Size"的文本文件以供R软件调用。

两项实验的数据集均符合响应变量的二项分布（binomial distribution）误差假设。本研究通过剔除无统计学意义的变量（P>0.05）筛选最优简约模型；若多水平变量（如体型或处理组）存在显著差异，则通过事后对比分析确定各水平间的差异。我们对两个简约模型均进行了误差分布与数据过度离散检验。

图3与图4ab均使用Excel软件，基于原始计数或计算得到的均值与标准误生成。支撑图3、图4a与图4b的原始数据存储于数据集文件"Dataset_Avian predators avoid attacking fly-mimicking"中同名的数据表（图3、图4a、图4b）中，该数据表同时包含绘图所用的公式与数据。双击图表即可查看数据生成图表的具体方式。生成的图表被导入PowerPoint软件，以添加标注文字与复制品照片；随后将幻灯片转换为JPEG（JPEG）格式文件。