Manuscript abstract: Herbivores may facilitate or impede exotic plant invasion, depending on their direct and indirect interactions with exotic plants relative to co-occuring natives. However, previous studies investigating direct effects have mostly used pairwise native-exotic comparisons with few enemies, reached conflicting conclusions, and largely overlooked indirect interactions such as apparent competition. Here we ask whether native and exotic plants differ in their interactions with invertebrate herbivores. We manipulate and measure plant-herbivore and plant-soil biota interactions in 160 experimental mesocosm communities to test several invasion hypotheses. We find that compared with natives, exotic plants support higher herbivore diversity and biomass, and experience larger proportional biomass reductions from herbivory, regardless of whether specialist soil biota are present. Yet, exotics consistently dominate community biomass, likely due to their fast growth rates rather than strong potential to exert apparent competition on neighbors. We conclude that polyphagous invertebrate herbivores are unlikely to play significant direct or indirect roles in mediating plant invasions, especially for fast-growing exotic plants.

Data abstract: We established 160 experimental ecosystems (mesocosm communities), manipulated interactions between plants, invertebrate herbivores and soil biota in a fully factorial design. Each mesocosm was grown in a 125 L pot (575 mm diameter), and comprised one of 20 unique, eight-species plant communities varying orthogonally in the proportion of exotic and woody shrub/tree species (0-100% and 0-63%, respectively). These plants were taken from a pool of 20 exotic and 19 native/endemic New Zealand plant species. Soil biota were manipulated using a modified plant-soil feedback approach, where each plant species was grown in monoculture in 10 L pots containing field-collected soil for 9-10 months, allowing the conditioning of typical associated soil biota for each of the plant species. We created ‘home’ soils by taking the conditioned soil from each of the eight representative species in a mesocosm and mixing it together to create a single inoculum. Each ‘home’ soil mixture was also used as an ‘away soil’ in a different mesocosm that did not contain any of the representative plants in that inoculum. These soils were intended to increase the relative biomass in inocula of specialized and preferred interaction partners of the resident (or non-resident) plant species, in a way that would mimic soils associated with established plant invasions. Invertebrate herbivore populations were added into half of the mesocosms with home soils and half with away soils. Thirteen invertebrate herbivore species introduced into the mesocosms successfully established, along with seven self-colonizing species, totaling 20 species in all. All mesocosms were sealed with mesh cages (15% shade factor) designed to retain added herbivores and exclude others from entering.

Additional details of the methods can be found in the supplementary materials of Waller et al. 2020: science.sciencemag.org/content/368/6494/967/suppl/DC1 and Allen et al. 2021: Nature Communications, in press.

 

Soil conditioning

The soils to be used in the larger mesocosm experiment were initially conditioned in the glasshouse by each of our focal species in order to create unique 1) ‘home’ soil inocula that would contain the suite of specialist and generalist soil biota cultured by the resident species growing in a given community and 2) ‘away’ soil inocula that would contain soil organisms not typically associated with the resident plants in a community. These soils could be practically interpreted as ‘previously-invaded’ vs. ‘uninvaded’. We grew 12-20 replicates of each individual plant species in 10 L pots containing live, field-collected soil mixed in equal parts with pasteurized field-collected soil and pasteurized sand. The soil was collected in late June and early July of 2016 from twelve subalpine, grass and shrub dominated sites located across western Canterbury and southern Marlborough. We chose sites where subsets of our focal plant species were present. At each site, we located three sampling areas that were representative of the site, but differed slightly in species composition, so that our final soil sample included rhizosphere soil from as many of our focal species as possible. At each sampling area, we used shovels to excavate chunks of sod measuring approximately 1 x 0.5 x 0.2 m, retaining as much loose soil that had fallen off the sod as possible. Soil and sod were stored outdoors for two weeks in weatherproof bags where temperatures averaged 1/11^o C (low/high). Aboveground vegetation was removed from sod chunks and soil and roots were passed through (2.5 cm²) sieves in mid-July to remove rocks and other debris, but retain any roots. This sampling provided us with approximately 375 L of soil from each site, which was mixed together in equal parts for a total of approximately 3000 L of inoculum. We also collected 3000 L of soil from a field on the Lincoln University campus for pasteurization in mid-July of 2016. Sand was collected from the Rakaia River valley, washed and sieved. The soil collected from the Lincoln site was mixed with the sand in equal parts and pasteurized in 500 L batches on a modified trailer bed fitted with steam pipes below a metal sheet and covered with a large tarpaulin. Each batch was brought up to a temperature of at least 100^oC, held at temperature for 60 minutes and cooled for 24 hours, before being treated a second time. Live soil was mixed with pasteurized soil in a 1:2 ratio.

After seeds had germinated and seedlings had at least one true leaf, they were transplanted into their own 1 L pot containing the soil inoculum. Some seedlings were too small to transplant directly into the large pot at this stage, so they were potted into a 500 mL pot of live inoculum until they were strong enough to go into the larger pot.  Plants were added to pots beginning in July 2016 and grew for approximately 9-10 months.

 

Mesocosm experimental design

Our mesocosm experiment incorporated a fully factorial design, with 20 plant communities X soil manipulation (home/away) X herbivore manipulation (herbivores added/not added). To begin, we designed 20 unique plant communities, each containing one individual of each of eight species, varying orthogonally in their proportion of exotic species (0, 25, 50, 75, 100%) and their proportion of woody species (0, 25, 38, 63%). Although we initially included seven nitrogen (N)-fixing exotic species, two species (one woody and one herbaceous) had extremely low survival, so effectively there were only five exotic N-fixers overall. Home soils contained a mixture of conditioned soils from each of the eight species occurring in that community, whereas away soils contained a mixture of conditioned soils from eight species occurring in one of the other 19 communities, but where a focal species did not occur. Twenty herbivore species established across the +Herbivore mesocosms (n = 80) and mesh cages were secured over each pot to ensure the insects remained in their pots. Seven of these herbivore species colonized from outside of cages (i.e. slugs and aphids) and we controlled these additions in -Herbivore but not +Herbivore mesocosms, where we allowed them to maintain populations. Thus, we consider our herbivore treatment to be ‘herbivores added’ vs. ‘herbivores reduced’.

 

Mesocosm establishment

The 160 mesocosms were established in a field on the campus of Lincoln University in Lincoln, New Zealand. We established the mesocosm communities in two phases. First, we germinated all of our plant species a second time, using the same method as described above. This time, however, after germination and two true leaves emerged, we planted each of our species into small pots containing their respective mesocosm soil inoculum before adding them to the mesocosms. This was done so the plants could be “hardened off” in treated soil before planting outdoors. To accomplish this, we harvested four pots of each plant species grown in the soil-conditioning phase, combining all soil and roots into a single bag for each individual species. We simultaneously pasteurized another batch of field-collected background soil from our field at Lincoln University to double this inoculum volume. Each seedling for the mesocosm phase was then allocated to a particular community and treatment combination: plants that were to go into home soil communities were planted in individual pots in soil conditioned by themselves, and seedlings to grow in away communities were planted in soil from one randomly chosen species from their predetermined away mixture. These seedlings grew for approximately one month and were moved outside to a shade house during their last week in the small pots. While seedlings were hardening off, we harvested the rest of the plants from the conditioning phase as before and created home and away soil inoculum mixtures that would go into each mesocosm pot. Finally, to complete the planting, we constructed steel pots (575 mm diameter), filled each with a bottom layer of 22 L of crushed gravel, then 88 L of pasteurized soil:sand, and finally 12 L of either home or away soil inoculum, mixed uniformly across the top. We mixed pasteurized soil with sand to improve the drainage in the soil. Seedlings were planted in a ring, equally spaced around the center of the pot in March of 2017.

 

Herbivore cage design

Herbivore cages were constructed using Cropsafe Protection Mesh (0.58 mm, 15% shade factor) from Cosio Industries (Auckland, New Zealand), designed to keep out small insects like aphids and psyllids. The mesh was cut and sewn into tubes (255 cm long, 81 cm diameter) with Dabond 25/V92 UV-resistant thread from Coats Industrial (Auckland, New Zealand). The tube shape was reinforced by threading No. 8 wire (4 mm diameter) through loops sewn 75 cm from the top and bottom of the mesh. One open end of each tube was tightly drawn together and closed with cable ties, then hung from an overhead wire. The open bottom of each cage was secured around the mesocosm pot with two bungee cords that were later replaced by 10 cm wide strips of steel closed with a bolt. For access to the mesocosm community, we cut a 50 cm vertical slit in one side of the cage that was closed by tightly folding the mesh over on itself and secured with three 50 mm foldback binder clips.

 

Herbivore addition

Herbivore populations were deliberately established in 80 mesocosms. Thirteen herbivore species that were added successfully established, along with seven self-colonizing species, totaling 20 different species (establishment success and other herbivore species characteristics are detailed in Supplementary Table 25 of Allen et al. 2021, Nature Communications). These species were all polyphagous or oligophagous and included 7 native and 13 exotic herbivores from multiple feeding guilds (leaf and root chewers, suckers, and miners). Each herbivore species was added to all +Herbivore mesocosms in equal density, regardless of whether a known host plant was present. Herbivore additions were staggered depending upon availability and some species were added multiple times to increase probability of establishment success and maintain populations (see Supplementary Methods of Allen et al. 2021, Nature Communications, for detailed description of protocols for each herbivore species). All self-colonizing species were regularly removed from -Herbivore mesocosms, including spillover from intentional additions, but were allowed to establish populations in +Herbivore mesocosms. Several of the herbivore species produced multiple generations in the mesocosm communities (i.e., multiple life stages observed, or more individuals observed than were introduced), such as leafrollers, aphids, leafhoppers, and slugs. Overall, our goal was not to replicate natural plant-herbivore communities, but to capture how native and exotic plants interact with a consistent suite of herbivores in novel communities, the preference and performance of the herbivores, and potential consequences for indirect effects.

 

Herbivore data collection

We measured herbivore richness, biomass, and leaf damage by chewing and scraping herbivores. Herbivores were surveyed on eight occasions: May, June, July, August, September and November in 2017 and January and April in 2018. For each survey, we counted the number of individuals of each herbivore species that were observed feeding on each plant. For species that reached high densities (e.g., aphids), abundance was estimated by surveying a portion of the plant and extrapolating to the entire plant. For some highly mobile or belowground herbivores it was difficult to reliably characterize feeding interactions through direct observation. For these species, we used restriction fragment length polymorphism (RFLP) to identify host plants with DNA extracted from frass, regurgitate, or gut contents (see below). Finally, because we could not practically measure the biomass of each individual herbivore from each mesocosm, we converted raw abundances to a standardized estimate of herbivore biomass for each species using mean dry biomass of a random sample of 10 individuals. To calculate the mean biomass of each herbivore species for each individual plant, we multiplied the total abundance of the herbivore by its mean dry biomass per individual, and then divided by the number of times that plant was surveyed (plants that died were surveyed less than eight times). To estimate total mesocosm herbivore biomass, we multiplied the mean dry biomass per individual for each herbivore species with its total abundance across all surveys, and then summed across all herbivore species.

For each survey, we also assessed leaf damage by chewing and scraping herbivores on each plant against six different categories (0 = no damage, 1 = 1-5% leaf area chewed or scraped, 2 = 6-25%, 3 = 26-50%, 4 =51-75%, 5 = >75%). We used these categories because of the large number of plants to survey and the difficulties of non-destructively measuring percent leaf area removal at finer resolution in situ. We obtained an overall estimate of damage throughout the experiment by transforming the categories to median percent damage values (e.g., category 3 = 38%) and calculating mean percent damage per survey for each plant.

 

Herbivore molecular diet analyses

For several highly mobile or belowground herbivore species, it was difficult or impossible to reliably characterize feeding interactions through direct observation. For these species, we used restriction fragment length polymorphism (RFLP) to identify host plants, using DNA extracted from frass, regurgitate, or gut contents. RFLP was considered well suited as a technique, given the low diversity of potential host plants (maximum of eight species, all of known identity), and allowed rapid and inexpensive identification of host plants. These molecular data were treated as any other observed plant-herbivore interaction, with the number of herbivore individuals observed to contain DNA of a given plant species weighted by mean biomass per individual and incorporated into the calculation of cumulative herbivore biomass for each individual plant and mesocosm. RFLP uses restriction enzymes to cut amplified DNA at enzyme-specific cutting sites and produce different sized DNA fragments that can be used to distinguish among genotypes, species, or broader taxonomic groups. By incubating samples overnight with up to three different restriction enzymes, we produced DNA fragment size combinations unique to the eight species in each of the 20 mesocosm communities, which were then visualized on agarose gel and cross-referenced against a database of known samples.

 

Mesocosm harvest and sampling

After one year of growth, we harvested all above and belowground plant material from each mesocosm community. Using spades, we carefully excavated each plant, disentangling roots of different species when necessary. Plants were bagged in the field and taken offsite to wash free of soil and other debris, then cut into root and shoot fractions. All shovels used for harvesting were scrubbed and rinsed in bleach for at least 10 minutes between mesocosms. Although the communities grew in pots for a year, there was no evidence that the plants were pot bound at the end of the experiment. The different plant species’ roots were easily separated from one another at harvest, and there was no root coiling around the pots whatsoever.

 

Additional data manipulations

We used normalized degree (i.e., the proportion of herbivore species that fed upon a given host plant out of the total herbivore species in the mesocosm) to quantify herbivore richness for each plant, because the number of invertebrate species that established varied among mesocosms. Measuring the plant-herbivore interactions of the entire community allowed us to estimate each species’ potential for apparent competition (PAC). PAC is a metric devised by Müller et al. (1999, Journal of Animal Ecology) that describes the sharing of interaction partners between two species in a community, and has been previously used to predict outcomes of indirect interactions in host-parasitoid communities. To estimate PAC for each host plant species pair in a given mesocosm, we calculated d_ij, the proportion of herbivore biomass attacking plant species i that is shared with plant species j. In the equation for pairwise PAC below, α represents link strength (i.e., herbivore biomass), i and j are the focal pair of host plant species, m is all plant species from 1 to H (the number of plant species in the community), k is a herbivore species, and l is all herbivore species from 1 to P (the number of herbivore species in the community).

                        (1)

After calculating pairwise PAC between all plants within each mesocosm, we quantified the potential for focal species i to exert apparent competitive effects (PAC_exerted) by summing PAC values for the focal species on all other community members (excluding intraspecific PAC; PAC = 0 if plants shared no herbivores). We also quantified the potential for focal species i to receive apparent competitive effects (PAC_received) by summing pairwise PAC values from all other community members to the focal plant. Because PAC should vary with the total number of herbivores in the community, but was calculated on a standardized scale within each mesocosm (i.e., using the relative strength of interactions), we weighted community-level PAC values using the total herbivore biomass of the focal plant (for PAC_exerted) or the rest of the community (for PAC_received).

Community: Each mesocosm was in one of 20 unique plant communities.

Mesocosm: Unique mesocosm number.

Soil.treatment: Home (H), Away (A).

Herbivore.treatment: Herbivores added to mesocosm (HERB) or not (NO_HERB).

Plant.number: Unique individual plant number.

Plant.species: Plant species name.

Herbivore.species: Herbivore species name.

Herbivore.provenance: Whether the herbivore is Native or Exotic to New Zealand.

Herbivore.reproduction.status: Whether the herbivore reproduced (Reproduced) in the mesocosm experiment or not (None).     

Herbivore.presence: Presence (1) or absence (0) of each herbivore species for each individual plant.

Mean.herbivore.biomass: Mean biomass (mg) of each herbivore species per survey occasion for each individual plant.

Above.plant.biomass: Shoot biomass (g) produced by the plant.

Below.plant.biomass: Root biomass (g) produced by the plant.

Total.plant.biomass: Total root and shoot biomass (g) produced by the plant.

Plant.normalized.degree: Proportion of herbivore species that were present with which the plant interacted.

Mean.damage: Mean damage (%) to plant leaf tissue from chewing and scraping herbivores per survey.

PAC.exerted: Potential for an individual plant to exert apparent competition on the rest of the community.

PAC.received: Potential for an individual plant to receive apparent competition from the rest of the community.

Prop.exotic.planted: Proportion of exotic plants (0.00, 0.25, 0.50, 0.75 or 1.00) planted in the community.

Prop.exotic.realised: Proportion of total mesocosm biomass made up by exotic plants at the conclusion of the experiment.

Above.plant.mesocosm.biomass: Total root and shoot biomass (g) produced in the community.

Below.plant.mesocosm.biomass: Total root biomass (g) produced in the community.

Total.plant.mesocosm.biomass: Total shoot biomass (g) produced in the community.

Total.exotic.herbivore.mesocosm.biomass: Total biomass (mg) of exotic herbivores produced in the community.

Total.native.herbivore.mesocosm.biomass: Total biomass (mg) of native herbivores produced in the community.

Total.herbivore.mesocosm.biomass: Total herbivore biomass (mg) produced in the community.

Mesocosm.herbivore.species.richness: Number of herbivore species that were observed feeding in the community over the duration of the experiment.

Mesocosm.mean.herbivore.damage: Mean damage (%) to leaf tissue from chewing and scraping herbivores per plant.

Plant.provenance: Whether the plant is Native or Exotic to New Zealand.

Functional.group: Plant functional group (Grass, Forb, Shrub, Tree).

Mycorrhizal.status: Plant mycorrhizal status (EMF = ectomycorrhiza, AMF = arbuscular mycorrhiza, NM = non-mycorrhizal). Non-mycorrhizal plants are only presumed to be non-mycorrhizal, based on conspecific and con-familial accounts of mycorrhizal status.

Nitrogen.fixing.status: Nitrogen fixing status of plant (Rhizobia, Frankia, No).

Specific.leaf.area: Mean specific leaf area (cm².g⁻¹) measured from local plant populations.