DOI: 10.5061/dryad.wwpzgmsgb
title: Achieving Bio-Protection in New Zealand Ecosystems mesocosm data

description: We established 160 experimental ecosystems (mesocosms), manipulated
 interactions between plants, soil biota and invertebrate herbivores in a
 fully factorial design. Each mesocosm was grown in a 125 L pot (575 mm
 diameter, Fig. 1B), 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. Invertebrate
 insect 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. In each mesocosm, we measured
 distinct ecosystem properties and processes that are relevant to carbon
 cycling dynamics: above and belowground biomass; total in situ soil
 respiration; basal respiration; microbial biomass (measured as
 substrate-induced respiration); decomposition (rate of standardized
 substrate mass loss); soil organic matter; nitrogen availability;
 herbivore biomass; arbuscular mycorrhizal and other fungal biomass (using
 neutral- and phospho-lipid fatty acid [NLFA and PLFA] biomarkers,
 respectively); and bacterial biomass (PLFA).
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
 <strong>Soil conditioning</strong>
 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 (details in table S3). We
 chose sites where subsets of our focal plant species (table S2) 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. 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. 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.
 <strong>Mesocosm experimental
 design</strong> 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 (table S1), 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 (table S4). Twenty herbivore species
 established across the +Herbivore mesocosms (n = 80) and mesh cages (see
 fig. 1A) 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’. <strong>Mesocosm
 establishment </strong> 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 (table S1) and treatment combination (table S4): 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. <strong>Mesocosm harvest and
 sampling</strong> 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. After all plants
 were removed, soil was homogenized by turning over for several minutes
 with the shovel, after which time a soil sample was extracted and taken
 into the laboratory. There, approximately 50 g of soil was passed through
 a 4 mm sieve, and stored at 4<sup>o</sup>C until processed
 (i.e. substrate induced respiration, basal respiration). Ten grams of soil
 was freeze dried for PLFA/NLFA analysis, 50 g dried and ground for
 nutrient analyses, 2.5 g added to 96 well plates from the Mo-Bio Power
 Soil Extraction Kit, and 10 g frozen at -80<sup>o</sup>C for
 archiving. In the laboratory, a small subsample of root
 was taken from each individual plant by taking approximately ten fine-root
 fragments from random places on the root ball, which were then bundled
 together, and a 1 cm cross section cut from the bundle using a sterile
 razor blade. This sample was placed into a 96 well plate from the Mo-Bio
 Power Soil Extraction Kit and frozen at -80<sup>o</sup>C as
 soon as a plate was full. After this sampling, all plant material was
 dried at 65<sup>o</sup>C and weighed.
 <strong>Decomposition
 (percentLoss):</strong> We measured the rate of a
 standard substrate lost over time using buried tea bags that were placed
 into each mesocosm at the initiation of the experiment and removed at
 harvest (approximately 12 months later). We used Harney & Sons
 Black English Breakfast tea bags. These bags were chosen because the tea
 bag material is plastic and did not degrade in the soil. Our goal was to
 use a standard substrate across all mesocosms that was equally novel to
 each mesocosm to avoid any home field advantage in decomposition. Camellia
 sinensis, being a member of the Ericales, was sufficiently distinct from
 any species in the mesocosms that the phylogenetic similarity would be low
 across all mesocosms. Tea bags were oven dried at
 65<sup>o</sup>C and weighed immediately before adding to the
 mesocosms. We added two tea bags to the center of each mesocosm, spaced
 150 mm apart. At harvest, bags were shaken free of soil, oven dried again
 and weighed. <strong>NLFAamf</strong>: AMF
 biomass as measured by 16.1ω5 NLFA biomarkers
 <strong>Bacterial.biomass</strong>: Bacterial biomass
 as measured by PLFA biomarkers
 <strong>X18.2w6c</strong>: Non-AM fungal biomass as
 measured by 18:2ω6 PLFA biomarkers
 <strong>GramNegative</strong>: Biomass of
 gram-negative bacteria, grouped using iso and anteiso saturated branched
 PLFAs <strong>GramPositive</strong>:
 Biomass of gram-negative bacteria, grouped using monounsaturated and
 cyclopropyl 17:0 and 19:0 PLFAs <strong>tC:
 </strong>total carbon<strong>; AN_kg.ha:
 a</strong>vailable nitrogen<strong>; OM:
 </strong>organic matter<strong>; CN:
 </strong>carbon:nitrogen<strong> </strong>
 Total carbon and nitrogen in dried soil material were analyzed
 using the Dumas type combustion process with an Elementar Vario-Max CN
 Elemental Analyzer. The sample was combusted at 900°C in an oxygen
 atmosphere. The combustion process converts any elemental carbon and
 nitrogen into CO<sub>2</sub>, N<sub>2</sub> and
 NO<sub>x</sub>. The NO<sub>x</sub> species were
 subsequently reduced to N<sub>2</sub>. These gases were then
 passed through a thermal conductivity cell to determine
 CO<sub>2</sub> and N<sub>2</sub> concentrations
 and the percentages of C and N calculated from the sample
 weights. <strong>CWSLA and CWSRL</strong>:
 Community-weighted specific leaf area and specific root length
 To calculate community-weighted SLA and SRL for each mesocosm, we
 multiplied each species’ mean trait value by its relative aboveground
 biomass in the mesocosm and then summed these values across all species in
 each mesocosm community.  <strong>R10:
 t</strong>otal respiration: Total soil
 respiration was measured in situ from each mesocosm pot using a PPSystems
 EGM-4 infrared gas analyzer (IRGA). In October 2017, a single PVC collar
 (50 mm deep, 103 mm diameter) was carefully inserted into the soil in the
 center of each pot so that 1-2 cm of the ring remained above the surface.
 Rings were left undisturbed throughout the course of the experiment.
 Surface CO<sub>2</sub> efflux (R10), soil temperature and
 moisture were measured during peak growth in December 2017 and again at
 harvest in April 2018. Pots were well-watered ahead of the measurements to
 ensure they were all at approximately similar moisture levels. Total soil
 respiration (R10) was calculated using the equation: R10 =
 Flux/EXP(308.56*((1/56.02)-((1/(Ts+273.15-227.13))))), where flux=the
 measured CO<sub>2</sub> efflux in
 µmol/m<sup>2</sup>/s and Ts is the soil temperature
 (<sup>o</sup>C). <strong>Basal
 respiration: </strong><strong> </strong>
 Basal respiration was measured as the amount of
 CO<sub>2</sub> evolved from a 130 mL bottle containing 10 g
 dry weight equivalent of soil at 55% water holding capacity over a 3 h
 period, at 25°C. Carbon dioxide concentrations were measured by injecting
 1 mL gas samples into a LiCor LI-7000 CO<sub>2</sub> analyser
 1 and 3 h after capping.
 <strong>Substrate-induced
 respiration</strong>: Substrate-induced
 respiration (a measure of microbial biomass) was measured in the same way
 as basal respiration, except that 0.1 g of glucose was added to each
 bottle before capping
description: Key to headings in data spreadsheet: 
 <strong>Mesocosm</strong>: Unique mesocosm
 number <strong>Soil.treatment</strong>:
 Home/Away/Sterile 
 <strong>Invasion.treatment</strong>: Invaded with Cytisus
 scoparius or uninvaded 
 <strong>Herbivore.treatment</strong>: Herbivores added (HERB)
 or not (NO_HERB) 
 <strong>Plant.community</strong>: Each mesocosm was in one of
 20 unique plant communities 
 <strong>Soil.community</strong>: Denotes the 'away'
 soil community the mesocosm was grown in 
 <strong>prop.exotic</strong>: Proportion of exotic plants (0,
 25, 50, 75 or 100%) planted in the community 
 <strong>prop.woody</strong>: Proportion of woody plants (0,
 25, 38 or 63%) planted in the community 
 <strong>Above, below, total biomass</strong>: Total shoot,
 root or root and shoot biomass produced in the community
 <strong>prop.Nfix</strong>: Proportion of N-fixing
 plant species planted in the community 
 <strong>Herbivore.biomass</strong>: Total herbivore biomass
 (mg) produced in the mesocosm 
 <strong>percentLoss</strong>: percent of standard substrate
 lost over the course of the experiment 
 <strong>NLFAamf</strong>: AMF biomass as measured by 16.1ω5
 NLFA biomarkers 
 <strong>Bacterial.biomass</strong>: Bacterial biomass as
 measured by PLFA biomarkers 
 <strong>X18.2w6c</strong>: Non-AM fungal biomass as measured
 by 18:2ω6 PLFA biomarkers 
 <strong>GramNegative</strong>: Biomass of gram-negative
 bacteria, grouped using iso and anteiso saturated branched
 PLFAs <strong>GramPositive</strong>:
 Biomass of gram-negative bacteria, grouped using monounsaturated and
 cyclopropyl 17:0 and 19:0 PLFAs <strong>tC:
 </strong>total carbon<strong>; AN_kg.ha:
 a</strong>vailable nitrogen<strong>; OM:
 </strong>organic matter<strong>; CN:
 </strong>carbon:nitrogen<strong> </strong> 
 <strong>CWSLA and CWSRL</strong>:
 Community-weighted specific leaf area and specific root length
 <strong>R10: t</strong>otal respiration
 <strong>Basal.Respiration: </strong>basal
 respiration <strong>SIR</strong>:
 Microbial biomass