Leaf gas exchange in Ipomopsis aggregata under manipulated snowmelt timing and summer precipitation

Vegetative traits of plants can respond directly to changes in the environment, such as those occurring under climate change. That phenotypic plasticity could be adaptive, maladaptive, or neutral.

We manipulated the timing of spring snowmelt and amount of summer precipitation in factorial combination and examined the responses of photosynthetic rate, stomatal conductance, and intrinsic water-use efficiency (iWUE) in the subalpine herb Ipomopsis aggregata. The experiment was repeated in three years differing in natural timing of snowmelt.

A 50% reduction in summer precipitation reduced stomatal conductance and iWUE. Combining natural and experimental variation, earlier snowmelt reduced soil moisture, photosynthetic rate and stomatal conductance, and increased iWUE.

Earlier snowmelt is a strong signal of climate change and can change expression of leaf morphology and gas exchange traits, just as reduced precipitation can. Stomatal conductance showed adaptive plasticity under some conditions.

Methods
We established an experimental manipulation of summer precipitation and snowmelt and then measured floral traits over three years, 2018 - 2020. We used a replicated split‐plot design, with snowmelt manipulated at the plot level and precipitation manipulated at the subplot level. The treatments were applied to the same plots each year. Six 7 m ⨉ 7 m plots were established within a 45 m ⨉ 25 m area of Maxfield Meadow, Gothic, CO, USA and three were randomly assigned early snowmelt treatments; a black 55% woven shade cloth was applied over the entire plot in the spring to accelerate snowmelt. Cloths were set out during spring melt off when snow height reached an average of 100 cm across the study site, monitored, and removed right after bare ground became visible. In 2019, a large avalanche ran through the site and deposited snow and debris, resulting in a later deployment and removal of shade cloth in two plots. The 2019 avalanche added snow that prevented early snowmelt in one plot, so for analysis we recoded it as having normal snowmelt timing.

Within each of the six snowmelt plots, four 2 m ⨉ 2 m subplots arranged in a square were randomly assigned one of four summer precipitation treatments. First, a water addition treatment simulated doubled summer precipitation based on the historical average in July from 1989 - 2006 measured at the EPA CASTNET weather station GTH161, 0.9 km northeast of Maxfield Meadow (www3.epa.gov/castnet/site_pages/GTH161.html). We added 14 L of tap water evenly to each 4 m2 subplot every 2 days to supplement daily precipitation by 1.75 mm. Second, a water reduction treatment intercepted approximately 50% of incoming precipitation using a half-covered 2 m × 2 m rainout shelter. The rainout shelters were constructed with a PVC pipe skeleton, with sloping clear corrugated plastic greenhouse roofing slats spaced evenly on top to cover half of the plot's surface area. Intercepted rainwater ran down these slats into an attached gutter, which then fed into a bucket on the ground. The shelter frames were camouflaged with green and brown paint to avoid deterring or attracting pollinators and herbivores. Third, mock rainout treatments controlled for any effects of the physical PVC structures but lacked slats to intercept rain. Fourth, control subplots were unmanipulated and received ambient rainfall. 

We measured leaf-level gas exchange on non-flowering plants on 5 - 8 days each year for a total of 315 measurements of 275 unique plants. Measurements were taken the following number of days after the average unmanipulated snowmelt plot melted: 2018: 47 - 78, 2019: 33 - 60, 2020: 45 - 94. Each day we took measurements from subplots in random order and measured the longest leaf on one haphazardly selected rosette per subplot that had not been previously measured that year and had a leaf longer than 25 mm. Two consecutive measurements were recorded per leaf and averaged. We excluded measurements where the estimated intercellular CO2 concentrations or photosynthetic rates were negative. Leaf gas exchange was measured using a Li-Cor 6400 XT Portable Photosynthesis system (Licor, Lincoln, Nebraska, USA). All leaf gas exchange measurements taken between 08:00 to 12:00 with saturating light conditions (PAR = 1800 μmol m-2 s-1), a leaf temperature of 27 °C, and a sample CO2 concentration of 400 ppm, following Wu and Campbell (2007). Gas fluxes were calculated by dividing by the leaf area inside the leaf chamber, measured in ImageJ (National Institute of Health, Bethesda, Maryland, USA). Each value reported is a mean across the plants measured in that subplot and year.

植物的营养性状可直接响应环境变化，例如气候变化背景下发生的环境改变。这种表型可塑性（phenotypic plasticity）可分为适应性、适应不良性与中性三种类型。

本研究采用析因设计操控春季融雪时间与夏季降水量，探究亚高山草本植物聚花吉利草（Ipomopsis aggregata）的光合速率、气孔导度与内在水分利用效率（intrinsic water-use efficiency, iWUE）的响应特征。实验在自然融雪时间存在差异的三个年度内重复开展。

夏季降水量减少50%会降低气孔导度与内在水分利用效率。结合自然变异与实验操控的结果来看，提前融雪会降低土壤含水量、光合速率与气孔导度，同时提升内在水分利用效率。

提前融雪是气候变化的显著信号，可如同降水减少一般改变叶片形态与气体交换性状的表达模式。气孔导度在部分环境条件下表现出适应性表型可塑性。

## 实验方法
我们于2018-2020年三个年度内开展夏季降水与融雪的操控实验，并测定植株的花部性状。本研究采用重复裂区设计：融雪操控在样地水平开展，降水操控在副样地水平开展，且每年均对同一组样地施加处理。在美国科罗拉多州哥特市Maxfield Meadow的45 m × 25 m区域内，设置6个7 m × 7 m的样地，其中3个被随机分配至提前融雪处理组：春季时向整个样地覆盖黑色55%遮光率的编织遮阳布，以加速融雪。遮阳布于春季融雪期布设，当研究区域内积雪平均高度达到100 cm时开始布置，待样地完全裸露后即刻移除。2019年，一场大型雪崩途经研究区域并携带积雪与碎屑，导致两个样地的遮阳布布设与移除时间均延后。其中一个样地因雪崩带来的积雪覆盖未能实现提前融雪，因此在数据分析阶段将其重编码为正常融雪时序。

在6个融雪样地内，各设置4个呈方形排列的2 m × 2 m副样地，随机分配至4种夏季降水处理组。其一为增水处理组：基于Maxfield Meadow东北0.9 km处的EPA CASTNET气象站GTH161（www3.epa.gov/castnet/site_pages/GTH161.html）1989-2006年7月的历史平均降水量，模拟夏季降水量翻倍的处理，每2天向每个4 m²的副样地均匀施加14 L自来水，以使日降水量增加1.75 mm。其二为减水处理组：采用半覆盖式2 m × 2 m避雨棚拦截约50%的自然降水。避雨棚以PVC管材搭建骨架，顶部均匀铺设倾斜的透明波纹塑料温室顶板，覆盖副样地一半的表面积。截留的雨水沿顶板流入配套水槽，最终汇集至地面的储水桶中。避雨棚骨架经绿、棕两色涂装以作伪装，避免干扰传粉者与植食性动物的行为。其三为模拟避雨棚处理组：该处理仅保留PVC结构框架，不安装挡雨顶板，用于控制物理结构本身带来的非降水效应。其四为对照组副样地：不施加任何操控措施，接收自然降水。

本研究于每年的5~8个测定日对非开花植株开展叶片水平气体交换测量，累计完成275株独特植株的315次有效测量。测定时间设置为对照样地平均融雪后的天数：2018年为47~78天，2019年为33~60天，2020年为45~94天。每日按照随机顺序对副样地开展测定，每个副样地随机选取1株此前未在当年被测定过、且叶片长度超过25 mm的莲座状植株，测量其最长叶片的气体交换参数。每片叶片记录两次连续测量值并取平均值。剔除估算胞间CO₂浓度或光合速率为负值的测量数据。叶片气体交换采用Li-Cor 6400 XT便携式光合测定系统（Li-Cor, 美国内布拉斯加州林肯市）进行测定，测定时段统一设置为08:00至12:00，光照条件设置为饱和光强（光合有效辐射PAR=1800 μmol·m⁻²·s⁻¹），叶温控制为27 ℃，样品CO₂浓度设置为400 ppm，测定方法参照Wu和Campbell（2007）的标准流程。气体交换通量通过叶室内部的叶面积进行归一化计算，叶面积采用ImageJ软件（美国国立卫生研究院，马里兰州贝塞斯达市）进行测量。本研究报告的每一项数值均为对应副样地与年度内所有被测植株的平均值。