Woody plant secondary chemicals increase in response to abundant deer and arrival of invasive plants in suburban forests

Plants in suburban forests of eastern North America face the dual stressors of high white-tailed deer density and invasion by nonindigenous plants. The combination of chronic deer herbivory and strong competition from invasive plants could alter a plant’s stress- and defense-related secondary chemistry, especially for long-lived juvenile trees in the understory, but this has not been studied. We measured foliar total antioxidants,  phenolics, and flavonoids in juveniles of two native trees, Fraxinus pennsylvanica (green ash) and Fagus grandifolia (American beech), growing in six forests in the suburban landscape of central New Jersey, USA. The trees grew in experimental plots that had been subject for 2.5 years to factorial treatments of deer access/exclosure X addition/no addition of the nonindigenous invasive grass Microstegium vimineum (Japanese stiltgrass). As other hypothesized drivers of plant secondary chemistry, we also measured non-stiltgrass herb layer cover, light levels, and water availability. Univariate mixed model analysis of the deer and stiltgrass effects and multivariate structural equation modeling (SEM) of all variables showed that both greater stiltgrass cover and greater deer pressure induced antioxidants, phenolics, and flavonoids, with some variation between species. Deer were generally the stronger factor, and stiltgrass effects were most apparent at high stiltgrass density. SEM also revealed that soil dryness directly increased the chemicals; deer had additional positive, but indirect, effects via influence on the soil; in beech PAR positively affected flavonoids; and herb layer cover had no effect. Juvenile trees’ chemical defense/stress responses to deer and invasive plants can be protective, but also could have a physiological cost, with negative consequences for recruitment to the canopy. Ecological implications for species and their communities will depend on costs and benefits of stress/defense chemistry in the specific environmental context, particularly with respect to invasive plant competitiveness, extent of invasion, local deer density, and deer browse preferences.

Methods
Data were collected in six suburban forests in central New Jersey, USA, within 16 m2 plots, with half fenced to exclude deer. Variables measured for this study were:

- Proportion cover of all plants in the herb layer, scored in 10% intervals and measured in 16 0.25 m2 subplots per plot

- Ground-level photosynthetically active radiation (PAR), measured as the percent of full sun PAR, using five measurements per plot made with an AccuPAR ceptometer

- Soil water potential measured on two samples per plot with a benchtop WP4 meter

- A deer browse index for each plot, set to zero for fenced plots and assigned to one common value for all unfenced plots within one forest, based on the proportion of deer-browsed individuals in unfenced plots of five native plant taxa: Carya spp., F. grandifolia, Fraxinus pennsylvanica, Acer rubrum, and Rubus allegheniensis. These were included because they were sufficiently common in the forests’ understories to allow for one index applicable to all of the forests and because they were, in our sites, neither the most browsed species nor completely avoided by deer-browsed

In these same plots, leaf tissue was collected from Fagus grandifolia and Fraxinus pennsylvanica, dried at 50 deg C and used in chemical analysis of total antioxidants, total phenolics, and total flavonoids. 

All data were entered and managed in Excel data files, and statistical analysis was done in SAS for mixed model analysis and in R (piecewiseSEM package) for structural equation modeling

北美东部城郊森林中的本土植物面临白尾鹿高密度种群与外来植物入侵带来的双重胁迫压力。长期鹿类啃食与入侵植物引发的激烈竞争，可能会改变植物与胁迫响应、防御相关的次生代谢化学特征，尤其是林下多年生幼树，但这一现象尚未得到相关研究。本研究对美国新泽西州中部城郊6处森林中的两种本土乔木幼树——绿梣（Fraxinus pennsylvanica，green ash）与美国山毛榉（Fagus grandifolia，American beech）——的叶片总抗氧化剂、酚类与黄酮类物质进行了测定。这些幼树生长于经过2.5年双因素实验处理的样地中，处理组合为鹿类访问/围栏排除 × 外来入侵禾本科植物日本莠竹（Microstegium vimineum，Japanese stiltgrass）添加/不添加。除上述核心处理外，我们还测定了影响植物次生代谢化学特征的其他潜在驱动因子：非日本莠竹以外的草本层盖度、光照水平与土壤水分有效性。

针对鹿类与日本莠竹影响的单变量混合模型分析，以及包含所有变量的多变量结构方程模型（Structural Equation Modeling，SEM）结果显示：更高的日本莠竹盖度与更强的鹿类取食压力均会诱导抗氧化剂、酚类与黄酮类物质的合成，且不同物种间存在一定差异。鹿类的影响整体更为显著，而日本莠竹的效应在其盖度较高时更为突出。结构方程模型还揭示：土壤干燥度会直接提升上述次生代谢物质含量；鹿类通过影响土壤间接产生正向调控作用；在美国山毛榉中，光合有效辐射（PAR）对黄酮类物质含量具有正向影响；草本层盖度则无显著效应。

幼树对鹿类与入侵植物的化学防御/胁迫响应兼具保护作用，但也可能带来生理成本，对其向冠层树木的定植产生负面影响。该研究结果对物种及其群落的生态学意义，取决于特定环境背景下胁迫/防御化学物质的成本与收益，尤其与外来入侵植物的竞争力、入侵程度、当地鹿类种群密度以及鹿类取食偏好密切相关。

### 研究方法
本研究的数据采集于美国新泽西州中部的6处城郊森林，实验样地面积为16 m²，其中半数设置围栏以排除鹿类。本研究测定的变量包括：
1. 草本层所有植物的盖度比例：以10%为间隔分级，通过每个样地内16个0.25 m²的小样方测定得到数据；
2. 地表光合有效辐射（PAR）：以全光照PAR的百分比表示，使用AccuPAR ceptometer在每个样地内进行5次测定；
3. 土壤水势：每个样地采集2份样本，使用台式WP4水分测定仪（benchtop WP4 meter）进行检测；
4. 每个样地的鹿类取食指数：围栏样地的指数设为0；未围栏样地的指数统一设置为同一森林内所有未围栏样地的共同值，基于5种本土植物类群——山核桃属（Carya spp.）、美国山毛榉（F. grandifolia）、绿梣（Fraxinus pennsylvanica）、红花槭（Acer rubrum）、悬钩子属（Rubus allegheniensis）中被鹿类啃食个体的比例计算得出。选取这些类群的原因是：它们在研究区域的林下群落中分布足够常见，可用于构建统一的取食指数，且在本研究样地中，它们既非鹿类最偏好啃食的物种，也未被鹿类完全回避。

在上述相同样地中，我们采集了美国山毛榉与绿梣的叶片组织，将其置于50℃下烘干后用于总抗氧化剂、总酚类与总黄酮类物质的化学分析。

所有数据均通过Excel电子表格录入与管理，统计分析方面，单变量混合模型分析使用SAS软件完成，结构方程模型分析则使用R语言的piecewiseSEM包完成。