DOI: 10.5061/dryad.0vt4b8gzd
title: Exotic plants accumulate and share herbivores yet dominate communities via rapid growth

description: 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.
description: 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.  
 <strong>Soil conditioning</strong>
 <em>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<sup>o</sup> C
 (low/high). Aboveground vegetation was removed from sod chunks and soil
 and roots were passed through (2.5 cm<sup>2</sup>) 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.</em>
 <em>Each batch was brought up to a temperature of at least
 100<sup>o</sup>C, 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.</em>
 <em>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.</em>  
 <strong>Mesocosm experimental
 design</strong> <em>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’.</em>  
 <strong>Mesocosm establishment </strong>
 <em>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.</em>  
 <b>Herbivore cage design</b>
 <i>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.</i>
   <b>Herbivore
 addition</b> <i>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.</i>  
 <b>Herbivore data collection</b>
 <i>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. </i>
 <i>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 = &gt;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.</i>  
 <b>Herbivore molecular diet analyses</b>
 <i>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.</i>  
 <strong>Mesocosm harvest and
 sampling</strong> <em>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.</em>  
 <strong>Additional data
 manipulations</strong> <i>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<sub>ij</sub>, 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).</i>
 <i>                        (1)</i> <i>After calculating pairwise PAC between all plants within each mesocosm, we quantified the potential for focal species i to exert apparent competitive effects (</i><i>PAC<sub>exerted</sub>) 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<sub>received</sub>) 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<sub>exerted</sub>) or the rest of the community (for PAC<sub>received</sub>).</i>
description: <b>Community: </b>Each mesocosm was in one of 20
 unique plant communities. <b>Mesocosm:
 </b>Unique mesocosm number.
 <b>Soil.treatment: </b>Home (H), Away (A).
 <b>Herbivore.treatment: </b>Herbivores added to
 mesocosm (HERB) or not (NO_HERB).
 <b>Plant.number: </b>Unique individual plant
 number. <b>Plant.species: </b>Plant species
 name. <b>Herbivore.species: </b>Herbivore
 species name. <b>Herbivore.provenance:
 </b>Whether the herbivore is Native or Exotic to New
 Zealand. <b>Herbivore.reproduction.status:
 </b>Whether the herbivore reproduced (Reproduced) in the mesocosm
 experiment or not (None).<b>      </b>
 <b>Herbivore.presence: </b>Presence (1) or absence
 (0) of each herbivore species for each individual plant.
 <b>Mean.herbivore.biomass: </b>Mean biomass (mg) of
 each herbivore species per survey occasion for each individual
 plant. <b>Above.plant.biomass: </b>Shoot
 biomass (g) produced by the plant.
 <b>Below.plant.biomass: </b>Root biomass (g) produced
 by the plant. <b>Total.plant.biomass:
 </b>Total root and shoot biomass (g) produced by the
 plant. <b>Plant.normalized.degree:
 </b>Proportion of herbivore species that were present with which the
 plant interacted. <b>Mean.damage: </b>Mean
 damage (%) to plant leaf tissue from chewing and scraping herbivores per
 survey. <b>PAC.exerted: </b>Potential for
 an individual plant to exert apparent competition on the rest of the
 community. <b>PAC.received: </b>Potential
 for an individual plant to receive apparent competition from the rest of
 the community. <b>Prop.exotic.planted:
 </b>Proportion of exotic plants (0.00, 0.25, 0.50, 0.75 or 1.00)
 planted in the community.
 <b>Prop.exotic.realised: </b>Proportion of total
 mesocosm biomass made up by exotic plants at the conclusion of the
 experiment. <b>Above.plant.mesocosm.biomass:
 </b>Total root and shoot biomass (g) produced in the
 community. <b>Below.plant.mesocosm.biomass:
 </b>Total root biomass (g) produced in the community.
 <b>Total.plant.mesocosm.biomass: </b>Total shoot
 biomass (g) produced in the community.
 <b>Total.exotic.herbivore.mesocosm.biomass: </b>Total
 biomass (mg) of exotic herbivores produced in the community.
 <b>Total.native.herbivore.mesocosm.biomass: </b>Total
 biomass (mg) of native herbivores produced in the community.
 <b>Total.herbivore.mesocosm.biomass: </b>Total
 herbivore biomass (mg) produced in the community.
 <b>Mesocosm.herbivore.species.richness: </b>Number of
 herbivore species that were observed feeding in the community over the
 duration of the experiment.
 <b>Mesocosm.mean.herbivore.damage: </b>Mean damage
 (%) to leaf tissue from chewing and scraping herbivores per
 plant. <b>Plant.provenance: </b>Whether the
 plant is Native or Exotic to New Zealand.
 <b>Functional.group: </b>Plant functional group
 (Grass, Forb, Shrub, Tree).
 <b>Mycorrhizal.status: </b>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.
 <b>Nitrogen.fixing.status: </b>Nitrogen fixing status
 of plant (Rhizobia, Frankia, No).
 <b>Specific.leaf.area: </b>Mean specific
 leaf area (cm<sup>2</sup>.g<sup>−1</sup>) measured
 from local plant populations.