Study System. We performed a selective predator exclusion experiment on ten woody host plant species at Great Hollow Nature Preserve in New Fairfield, Connecticut, USA (41.507998 N, -73.530032 W). The preserve is 334 ha and comprised predominantly of mature, closed-canopy, second-growth deciduous and mixed forest. Historic disturbance of the land, mostly from past agricultural uses, has favored the establishment of many of the invasive plants that are now ubiquitous to the northeastern U.S. and often aggressively targeted for removal by land managers and conservation practitioners. We focused our experiment on a subset of these invasive plants: Japanese barberry (Berberis thunbergii), Morrow’s honeysuckle (Lonicera morrowii), burning bush (Eunonymous alatus), and autumn olive (Eleagnus umbellata). These four species are designated as invasive by the Connecticut Invasive Plants Council, formed via Connecticut General Statutes § 22a-381a through § 22a-381d (https://cipwg.uconn.edu/ipc/). For comparison, we chose six native woody plants that co-occur with these invasive shrubs and are the most dominant native trees in the understory of our study system: striped maple (Acer pennsylvanicum), shadbush (Amelanchier canadensis), musclewood (Carpinus caroliniana), witch-hazel (Hamamelis virginiana), sweet birch (Betula lenta), and American beech (Fagus grandifolia). Performing our experiment across these 10 species thus provided a community-wide perspective on the impacts of invasive plants on food webs, in the context in which invasive plant management decisions should be made (Westman 1990).
Bird exclusion experiment. From 4–27 May, 2021, we set up a predator exclusion experiment in a paired design following Singer et al. (2012). Briefly, insectivorous birds were prevented from foraging on branches of our 10 study species via mesh netting (Bird-X Protective Netting, Elmhurst, IL, USA) that was folded and sown into a bag that was slid over a single branch of a target plant, and affixed using plastic zip-ties (“exclusion treatment”). Each of these branches was paired with a nearby (< 10 m away) unmanipulated control branch of the same species. We set up 12 treatment pairs for each of the 10 focal plant species, resulting in a total of 240 individual host plants in the study. At the end of the set-up period on 27 May, all 240 branches were struck with a 0.3 m wooden dowel to dislodge arthropods and reset colonization to avoid bias caused by the disturbance of setting up the exclusion netting. After a 2-wk waiting period, we then sampled foliage-foraging arthropods with a branch-beating technique (Wagner 2005) every other week from 24 May until 2 July, to coincide with the peak breeding period of most forest birds in our region. We struck each branch with a 0.3 m dowel while held over a 1m2 ripstop fabric beat sheet and collected all invertebrates from the beat sheet into plastic vials or plastic zip-top bags using aspirators or soft-touch aluminum forceps. Each branch was sampled this way three times with 14 d between samples. We kept the collected arthropods cool in the field in coolers with ice packs and then transferred them to a -80º C freezer at the end of each day.
Taxonomic identification of arthropods. We combined the three repeated samples from a given branch to provide a tally of total arthropod abundance (Clark et al. 2016) and then weighed (wet mass) the arthropods together on a 10− 4 g microbalance. After identifying all invertebrates from a given branch to class, we sorted all insects in the orders Lepidoptera, Hemiptera, Hymenoptera to family. We identified true spiders (Araneae) and Opiliones to family as well. Following identification, we transferred each taxonomic group from a given branch to separate 0.6-2 mL Eppendorf tubes and stored them at -80º C. In all, the four numerically dominant taxonomic groupings of arthropods included (1) Lepidoptera (caterpillars), (2) true spiders (Araneae), (3) herbivorous Hemiptera families (Aphidae, Cicadellidae, Membracidae, Miridae, and Pentatomidae), and (4) Orthoptera (families Gryllidae and Tettigoniidae).
Elemental analysis of arthropods. As an indicator of arthropod quality as prey for songbirds, we used elemental analysis to compare the protein content (percent elemental Nitrogen) of arthropods collected from native plants and invasive plants (Smets et al. 2021). Protein is a macronutrient that strongly mediates food selection by breeding birds and is critical to offspring development (Klasing 1998, Birkhead et al. 1999, Robbins et al. 2005, Razeng and Watson 2015). Our preliminary analyses suggested that two broad functional groups responded strongly to bird predation effects and varied significantly among native and invasive host plants, each representing a different trophic level above host plants: foliage-feeding herbivores (see Online Resource 1, Fig S1-S3) and predatory true spiders (Araneae). These two groupings of arthropods are prey for foliage-gleaning, insectivorous birds, should differ in protein content because of their different trophic levels (Reeves et al. 2021), and are impacted by experimental manipulation of bird predation (Gunnarsson et al. 1996). Generally, insects feeding on plants have a similar C:N ratio as their host (Abbas et al. 2014). To assay elemental composition, we first pooled foliage-feeding herbivore taxa and true spiders across sampling periods for each branch in the bird exclusion treatment group. We limited our analyses to branches with birds excluded to quantify the nutritional quality of the arthropod community as it would be for the first bird foraging on a given branch. We then oven-dried arthropod samples at 60° C to a constant mass and homogenized any samples that weighed > 3 mg. Samples (1.5–3.5 mg) were measured for carbon and nitrogen concentrations on a Flash 1112 CHNSO elemental analyzer (CE Elantech inc. Lakewood, NJ, USA) by comparing results with aspartic acid and L-cystine standards. We analyzed replicates for a subset of branches, producing mean within-sample coefficients of variation of 4.2% for nitrogen and 2.9% for carbon.
Statistical analyses. We employed a series of Generalized Linear Mixed Models (GLMMs) using the lme4 package (Bates et al. 2015) in R version 4.1.2 (R Development Core Team, 2022). We included the following as response variables for successive models: (1) total arthropod biomass sampled per plant, (2) spider abundance (Araneae), (3) caterpillar abundance (Lepidoptera), (4) herbivorous true bug abundance (Hemiptera) (5) tree cricket and katydid abundance (Orthoptera) (6) N content of herbivorous insects and (7) N content of spiders. Arthropod biomass was fitted as a normally distributed GLMM after log-transformation and included both host plant species and bird exclusion treatment as fixed effects, and branch as a random effect. All abundance models were fitted with a negative binomial GLMM. In abundance models, invasive status (yes or no) was a fixed effect along with bird-exclusion treatment, and branch and host-plant species were included as random effects. Samples taken across the three sampling periods were pooled together in arthropod models to avoid pseudoreplication (Clark et al. 2016). Nitrogen content models were fit with a normal distribution, but since all arthropod samples were pooled across sampling periods and only taken from exclusion branches, only host-plant species was used as a main effect (GLM). Post-hoc tests comparing changes in biomass, abundance, and nitrogen content were run using the emmeans package in R (Lenth 2016). Differences were investigated between pooled native plants and each individual invasive plant using Dunnett’s method for P-value adjustment in unplanned contrasts. P-values and critical values were determined using the car package with analysis of deviance tests and χ2 test statistics (Fox et al 2015).
Log-response ratios. A follow-up GLM was employed using LRRs (log-response ratios) of exclusion treatments to investigate the interspecific variation in bird predation effects across all host plant species (Singer et al. 2012). LLRs, when used to evaluate the effects of natural enemy exclusion, provide insight into whether the interaction strength of top-down effects vary according to different environmental variables (Chaguaceda et al. 2021, Wooton 1997). In this case, we used a LRR modified from Hedges et al. (1999) as the natural log of the combined arthropod biomass on exclusion branches divided by the arthropod biomass on control branches. LLR calculated in this way tests the prediction that bird predation is weaker on invasive plants, testing the predictions of the ‘weaker predatory effects hypothesis’.