Study system

Triadica sebifera (Euphorbiaceae) is a rapidly growing tree native to Asia and invasive in the United States of America39. Triadica sebifera supports a diversity of herbivores in China (Supplementary Fig. 1)40. For example, Bikasha collaris (Coleoptera: Chrysomelidae) is a T. sebifera specialist flea beetle with leaf-feeding adults and root-feeding larvae41. Heterapoderopsis bicallosicollisis (Coleoptera: Attelabidae) is a specialist weevil with leaf-chewing adults, and larvae that feed in rolled leaf structures called nidi42. Lepidoptera whose caterpillars attack this tree include generalists such as Cnidocampa flavescens (Limacodidae), Grammodes geometrica (Noctuidae), Prodenia litura (Noctuidae) and Biston marginata (Geometridae) and the specialist Gadirtha inexacta (Noctuidae)43. In addition, generalist Aphis sp. (Homoptera: Aphididae) feed on T. sebifera phloem40. All these leaf-feeders are among the most abundant and damaging insects and commonly feed concurrently with belowground B. collaris larvae on T. sebifera from July to September in the Wuhan, China (Supplementary Fig. 1). Thus, these insects used in this experiment are most representative of the natural composition of herbivore assemblages on T. sebifera in the study area.

Plants and insects

We used T. sebifera seeds from a natural population near Wuhan, China (31°33′N, 114°07′E), for Experiments 1 and 2 and seeds from six populations across southern China and six populations across the southeastern United States for Experiment 3 (Supplementary Table 2). We planted seeds in an unheated greenhouse at Wuhan Botanical Garden, Hubei, China (30°32′N, 114°24′E). After 4 weeks, when plants had four or five leaves, we transplanted them individually into pots filled with topsoil (collected from a local field without T. sebifera) and grew them in an unheated greenhouse with ambient temperature and humidity and natural light until the start of each experiment.

For belowground herbivory, we obtained B. collaris eggs by having adults lay eggs in Petri dishes following Huang et al.28. For aboveground herbivores, we collected nidi of H. bicallosicollisis and larvae of G. inexacta and C. flavescens from a field in Wuhan, reared them on local T. sebifera plants under laboratory conditions (26–30 °C, 50–70% relative humidity, 14:10 h light : dark photoperiod), and used their offspring for experiments. We collected B. collaris adults and Aphis sp. aphids from sites in Wuhan for experiments.

Experiment 1: effects of aboveground B. collaris adults and heterospecific insects on belowground B. collaris larvae

To test the effects of conspecific and heterospecific aboveground herbivory on belowground B. collaris larval survival, we used aboveground treatments of damage by adults of B. collaris, H. bicallosicollisis and Aphis sp., and larvae of G. inexacta and C. flavescens, as well as mechanical damage and an undamaged control (Supplementary Fig. 1). The aboveground herbivores studied included representatives of three orders (Coleoptera (2 species), Lepidoptera (2) and Hemiptera (1)), two types of host ranges (specialist (3) and generalist (2)) and two feeding guilds (leaf chewer (4) and phloem feeder (1)).

Six weeks after transplantation (day 0), we selected similarly sized plants (height: 23.9±0.4 cm, stem diameter: 2.61±0.04 mm, number of leaves: 16.9±0.3) and transferred ten newly laid B. collaris eggs onto the roots of each plant following Huang et al.28. Then, we enclosed each plant in a nylon mesh cage (100 cm high, 20 cm diameter) and arranged plants randomly within eight rows in an unheated greenhouse. Within each row, we randomly assigned two plants to each of the seven aboveground herbivory treatments (2 replicates × 7 herbivory treatments, 14 plants in each row, 16 replicates in total). Rows were 1 m apart and plants within each row were 0.3 m apart from each other. Row did not explain any response variable and is not included in analyses.

To make aboveground and belowground herbivory occur simultaneously, we imposed aboveground herbivory treatments on day 9 when B. collaris eggs were hatching and left aboveground herbivores on the plants for 18 days, which is the average larval development time41. To make aboveground and belowground herbivory experimentally independent, we sealed the nylon mesh cage of each pot using string attached to the plant stem below all leaves. For conspecific aboveground herbivory, we added ten B. collaris adults to the leaves of each plant. Previous studies reported that ten adults removed approximately ten percent of the leaf area in 18 days when ten larvae of B. collaris attacked the plant simultaneously41. To reduce confounding effects of damage level on belowground B. collaris larval performance, we also selected ten percent of leaf area removed for the damage level of heterospecific aboveground herbivory treatments. For H. bicallosicollisis herbivory, we added two newly emerged male adults to each plant and replaced individuals that died during the experiment. For caterpillar herbivory, we added one first-instar G. inexacta larva or two first-instar C. flavescens larvae to each plant, because per capita feeding rate of G. inexacta is higher than that of C. flavescens44. We replaced larvae of G. inexacta and C. flavescens with new first-instar ones every 4 and 3 days, respectively, as late instars of both species can quickly cause severe damage, which would exceed the 10% target for defoliation. For herbivory by Aphis sp., we placed 50 adult aphids on each plant. This density is consistent with average abundance observed for the same size of aphids attacking plants in the field (51.7±3.1 aphids per plant, n=60). For mechanical damage, we punched seven to nine holes (diameter 0.6 cm) into randomly selected leaves of each plant every day. The quantity and timing of leaf area removed by mechanical damage coincided with real herbivory treatments. We did not manipulate control plants.

On day 27, we removed all aboveground herbivores and visually estimated the leaf area removed from each plant. There were no significant differences in leaf area removed among aboveground herbivory treatments (F 4,75 =1.22, P=0.31; one-way ANOVA, n=80; excluding aphid herbivory treatment and control). We harvested 8 of the 16 plants from each treatment and analysed condensed tannin, total tannin and total phenolic concentrations in the roots. We observed and removed B. collaris adults emerging from the soil on the remaining eight plants every day.

Experiment 2: field interactions between aboveground herbivores and belowground B. collaris larvae

To further examine the effects of conspecific and heterospecific aboveground herbivory on belowground B. collaris larval survival under natural conditions, as well as the effects of belowground B. collaris larvae herbivory on aboveground herbivore communities, we conducted a manipulative experiment in a field at Wuhan Botanical Garden. In this field, there was a 5-year-old T. sebifera stand (about 240 plants), with 1 m spacing between plants. Plants in the stand were heavily damaged by abundant B. collaris adults and had some damage from specialist, H. bicallosicollisis and G. inexacta, and generalist, C. flavescens and Aphis sp. The stand was surrounded by various vegetable crops and herbs (typical of environments where T. sebifera occurs naturally in China), which were attacked by generalist insects, including C. flavescens, G. geometrica, B. marginata, P. litura and Aphis sp. (Supplementary Fig. 1).

To examine the effects of B. collaris adult abundance on larval survival, we established nine blocks (1 × 1 m) in the field that varied in distance to the T. sebifera stand and were presumably exposed to a gradient of B. collaris adult abundance. Two blocks were 7 m apart in the centre of the T. sebifera stand colonized by B. collaris. Seven blocks were outside of the T. sebifera stand with each an additional 8 m away from the stand. Blocks were plowed and sprayed twice with herbicide before the start of the experiment. Ten weeks after, seedlings were transplanted (day 0), 20 pots with similarly sized plants (stem height: 31.0±0.3 cm, stem diameter: 3.48±0.04 mm, number of leaves: 29.4±0.4) were buried 15 cm deep in the soil in each block (five rows of four plants). We added belowground herbivores (ten newly laid B. collaris eggs per plant—see above) to ten randomly chosen pots in each block and left the other ten pots as controls (damaged plants neighbouring control ones). To prevent oviposition by adults into the soil of pots but allow the aboveground plant parts (leaves and stems) to be exposed to naturally occurring herbivores, we enclosed the belowground parts of each plant with mesh.

To examine the effects of belowground B. collaris larvae on conspecific and heterospecific aboveground herbivores, we visually checked all leaves on each plant and recorded the identity and abundance of insects on all plants (n=180) every 3 days over a period of 36 days, since belowground B. collaris development time including egg, larva and pupa is about 36 days41. Meanwhile, we also visually estimated leaf area damaged by B. collaris or caterpillars to the nearest 5% every 3 days. B. collaris adult feeding produces irregular pits and scars, whereas caterpillar feeding on leaves produces large irregular holes or skeletonized leaves. Damage made by H. bicallosicollisis is similar to that of B. collaris adults. We only found one H. bicallosicollisis during the field survey and so assumed all irregular pits and scars were due to B. collaris in our analyses. To determine the effects of conspecific and heterospecific aboveground herbivores on belowground B. collaris larvae, we counted the number of B. collaris adults emerging from the soil for plants that received belowground herbivores (n=90).

Experiment 3: phenolic compounds as the mediator of aboveground and belowground herbivores

To test whether changes in phenolic compounds might be the causal mechanism underlying aboveground and belowground herbivore interactions, we assayed phenolic compounds in leaves and roots of different T. sebifera ecotypes after attack by larvae and/or adults of B. collaris. Previous studies reported that plants from China have higher tannin levels than their US counterparts44,45,46. Therefore, this experiment was structured as a 2 × 2 × 12 full factorial design incorporating two levels of aboveground herbivory (0 versus 10 adults per plant), two levels of belowground herbivory (0 versus 10 eggs per plant), 12 T. sebifera populations (six per range, Supplementary Table 2) and six replicates.

Six weeks after transplantation (day 0), we selected similarly sized plants, enclosed each in a nylon mesh cage, and randomly assigned four plants of each population to each of six rows in an unheated greenhouse with the same spacing as in Experiment 1. Row did not explain any response variable and is not included in analyses. We randomly assigned one plant of each population in each row to one of the four different herbivory treatments. The timing of herbivory treatments was the same as in Experiment 1.

At the end of the herbivory treatment period (day 27), we harvested three plants of each treatment combination and analysed condensed tannin, total tannin and total phenolic concentrations in leaves and roots (three replicates). For remaining plants, we recorded the number of surviving B. collaris adults on plants subjected to aboveground herbivory treatments and removed them, and counted the number of adults emerging from the soil of plants with belowground herbivores daily (three replicates).

Chemical analyses

We dried leaves and roots of each plant separately at 40 °C for 5 days. We ground samples in a ball mill then stored them at −20 °C in sealed plastic bags before chemical analyses. We extracted samples (100 mg) with 1 ml of 70% (v/v) acetone (1 h, room temperature), sonicated them (20 min), and then, removed insoluble material by centrifugation (5 min, 10,000 r.p.m., 4 °C). We estimated condensed tannin content using the acid butanol assay45,47 by mixing leaf extracts (0.5 ml) with 6 ml of butanol-HCl reagent (n-butanol/concentrated HCl, 95:5, v/v) and 0.2 ml of iron reagent (2% FeNH 4 (SO 4 ) 2 ·12 H 2 O in 2 N HCl), oven heating mixtures (100 °C, 60 min), cooling them to room temperature and measuring their absorbance at 550 nm. We used commercial cyanidin chloride (Sigma-Aldrich) as a standard. We estimated total tannin content using a radial diffusion assay48 by placing leaf extracts (40 μl) in 5 mm diameter holes in 1% (wt/v) agarose plates with 0.1% (wt/v) BSA, incubating plates (30 °C, 96 h) and measuring precipitated protein area. We used commercial tannic acid (Sigma-Aldrich) as a standard. We estimated total phenolic content using the modified prussian blue assay49. We mixed sample extracts (100 μl) with 3 ml distilled water, 1 ml of 0.016 M K 3 Fe(CN) 6 and 1 ml of 0.02 M FeCl 3 . After 15.0 min, we added 5 ml of stabilizer (0.2% Gum Arabic in 17% H 3 PO 4 ) and measured absorbance at 700 nm. We used commercial Gallic Acid Monohydrate (Sigma-Aldrich) as a standard. All chemical concentrations are expressed as mg g−1 dry weight.

Data analyses

For Experiment 1, we used one-way ANOVAs to analyse the impacts of aboveground herbivores on belowground B. collaris larval survival and phenolic compounds in roots. We used Student–Newman–Keuls post-hoc tests for multiple comparisons among treatments.

For Experiment 2, we performed separate regression analyses to determine the relationships between belowground B. collaris larval survival and leaf area damaged by conspecific or heterospecific herbivory. We calculated leaf area damaged per plant by averaging across surveys. We analysed the impact of belowground B. collaris larval herbivory on the abundance and leaf area damaged by conspecific and heterospecific herbivores separately using repeated measures ANOVAs (fixed effect: larval herbivory, block; time: 11 level categorical variable).

For Experiment 3, we used two-way ANOVAs to analyse the impact of herbivory and plant origin on belowground B. collaris larval survival and root phenolic compounds (three fixed effects, origin, adult herbivory and their interaction), and on aboveground B. collaris adult survival and leaf phenolic compounds (three fixed effects, origin, larval herbivory and their interaction). Plant populations (six populations per range) were nested within origin as the random effect. We performed separate regression analyses to determine the relationship between insect survival and phenolic compounds.

To examine the role of changes in phenolic compounds in the effects of experimental treatments on survival in the ANOVAs, we performed regressions of survival on phenolic compounds and examined the dependence of regression residuals on experimental treatments in follow-up ANOVAs. In these analyses, significant effects in survival and chemical ANOVAs along with a significant regression result and a lack of a significant effect in the residual ANOVA indicates survival responses are strongly related to chemical changes.

We performed all data analyses with SAS, version 9.1 (SAS Institute).