Reduced seed set under water deficit is driven mainly by reduced flower numbers and not by changes in flower visitations and pollination

Water deficit can alter floral traits with cascading effects on flower-visitor interactions and plant fitness. Water stress induction can diminish productivity, directly resulting in lower flower production and consequently seed set. Changes in floral traits, such as floral scent or reward amount, may in turn alter pollinator visitations and behavior and consequently can reduce pollination services resulting in lower reproduction output. However, the relative contribution of this indirect in comparison to the direct effects of changes in seed set are not fully understood.

We manipulated water availability using rain-out shelters in a field experiment and measured effects on floral scent bouquet, morphology, phenology, flower-visitor interactions, pollination, and seed set. Plant individuals of Sinapis arvensis (Brassicaceae) were randomly assigned to one of three treatments: mean precipitation (= control), reduced mean precipitation, or drought period treatment.

Our results show that decreasing water availability lowers the number of flowers and seed set. This indicates a direct link between water stress and seed set, as seed mass increases with increasing flower number. The indirect link of water stress via floral traits, pollinator visits, and pollination has weaker effects on seed set. However, floral traits remain relatively stable under decreased water availability, whereas plant growth and flower abundance decrease, potentially in order to allow investment in more resources in fewer flowers to maintain pollination success. Thus, plants are able to compensate for water stress and can maintain floral trait expression, such as a stable scent emission and bouquet, to retain pollinator attraction.

These findings indicate that the direct link from water stress to seed set has a stronger impact on plants' reproductive success than the indirect link through altered floral trait expression and pollinator visits in a generalist plant species.

Methods
- manipulation of water availability on wild mustard (Sinapis arvensis) using rain-out shelters

- 3 treatments: well-watered, reduced watered, drought period; stem water potential was measured to conform plant's water stress and was used as the linear variable for further statistical analysis

- "Foral_traits"-file: following parameters were measured once in three freshly open flowers per plant from a low, middle, and high position to avoid position and age effects: stamen, style, and calyx length, petal length and width, flower display size, and nectar volume. On two flowers per plant, we collected one anther each in order to count pollen grains by means of a microscope (after 3 weeks under treatment). For pollen and nectar collection, nearly open flowers were covered with mesh bags the day before collection in order to prevent access by flower visitors. The inflorescence size (greatest expansion) was measured once on five inflorescences per plant. Floral height (height of the highest flower) was measured weekly with a folding yardstick; means values were included in the data file and were used for statistical analyses

- "Int_data"- file: plant-animal interactions were observed: each plant individual was observed daily; the number of visits by arthropods was recorded; the number of visits per day and per flower was calculated and used for further statistical analyses

- "Scent_data"-filechemical (= floral scent) data were analyzed using a thermal desorption system coupled with a GC-MS; compounds were analyzed and identified using GCMSsolution package; compounds in flowers were compared with those found on blank controls; the amount of compounds was estimated by comparing peak areas with area of a standard (Octadecane C18); absolute compound amounts included in the data file

- "Loggerdata"-file: raw data obtained from data loggers that were placed under each shelter; measured temperature and relative air humidity; one logger was placed next to the shelters and recorded temperature, relative air humidity, wind speed, photosynthetic active radiation PAR and air pressure

- "Soil_humi_temp"-file: raw data of weekly measured soil humidity and soil temperature next to each plant individual using avolumetrix water content sensor (Hydrosense II, Campbell Scientific) and a simple thermometer (DET3R, Voltcraft)

- "Plant_list"-file:  file with individual plant information

水分亏缺可改变花部性状，并对访花昆虫互作及植物适合度产生级联效应。水分胁迫会降低植物生产力，直接导致开花数量减少，进而降低结籽率。花部性状（如花香或报酬量）的改变，又会影响传粉者的访花行为与频次，最终削弱传粉服务，降低繁殖产出。然而，相较于结籽率变化的直接效应，此类间接效应的相对贡献尚未完全明晰。

本研究通过野外实验，利用降雨排除棚（rain-out shelters）调控水分可利用性，探究其对花香谱、花部形态、物候、访花昆虫互作、传粉效率及结籽率的影响。将田芥菜（Sinapis arvensis，十字花科Brassicaceae）的植株随机分为三组处理：正常降水组（对照组）、降水减少组、干旱周期处理组。

研究结果显示，水分可利用性降低会减少开花数量与结籽率，这表明水分胁迫与结籽率之间存在直接关联——因为种子质量随开花数量增加而提升。而水分胁迫通过花部性状、传粉者访花及传粉过程产生的间接关联，对结籽率的影响较弱。不过，在水分可利用性降低的条件下，花部性状相对稳定，而植物生长与开花丰度下降，这可能是为了将更多资源投入到少量花朵中，以维持传粉成功率。由此可见，植物能够对水分胁迫进行补偿，维持花部性状的表达（如稳定的花香释放与组分），从而保留对传粉者的吸引力。

本研究结果表明，在广适性植物物种中，水分胁迫直接影响结籽率的通路，对植物繁殖成功的影响强于通过改变花部性状与传粉者访花行为的间接通路。

### 方法
- 利用降雨排除棚（rain-out shelters）调控野生田芥菜（Sinapis arvensis）的水分可利用性
- 设置3种处理：正常供水组、减水处理组、干旱周期处理组；测定茎水势以确认植株的水分胁迫程度，并将其作为后续统计分析的线性变量
- "Floral_traits"数据集文件：为避免植株位置与花龄带来的误差，从每株植物的低、中、高三个位置各选取1朵刚开放的花，一次性测定以下参数：雄蕊长度、花柱长度、花萼长度、花瓣长宽、花展示面积以及花蜜量。另外从每株植物的2朵花中各采集1枚花药，通过显微镜计数花粉粒（处理3周后开展计数）。为避免访花昆虫干扰，在采集花粉与花蜜的前一日，将待采集的即将开放的花朵用网袋套住。每株植物选取5个花序，测定其花序最大扩张尺寸。每周使用折叠卷尺测定植株的花部高度（最高花朵的高度），将均值纳入数据文件并用于统计分析
- "Int_data"数据集文件：观测植物-动物互作情况：每日对每株植株进行观测，记录节肢动物的访花次数，计算每日每朵花的访花频次，用于后续统计分析
- "Scent_data"数据集文件：花香数据采用热脱附系统耦合气相色谱-质谱联用仪（GC-MS）进行分析；使用GCMSsolution软件包对化合物进行分析与鉴定；将花朵中的化合物与空白对照样本进行比对；通过将峰面积与标准品（十八烷C18）的峰面积对比，估算化合物的含量，数据文件中包含化合物的绝对含量
- "Loggerdata"数据集文件：原始数据来自放置于每个遮蔽棚下方的数据记录仪，测定温度与相对空气湿度；另外在遮蔽棚旁放置1台记录仪，记录温度、相对空气湿度、风速、光合有效辐射（PAR）以及大气压力
- "Soil_humi_temp"数据集文件：使用容积含水量传感器（Hydrosense II, Campbell Scientific）与简易温度计（DET3R, Voltcraft），每周测定每株植物旁的土壤湿度与土壤温度的原始数据
- "Plant_list"数据集文件：包含单株植株的相关信息