DOI: 10.5061/dryad.4xgxd257b
title: Biogeographic differences in plant-soil biota relationships contribute to the invasion exotic range expansion of Verbascum thapsus

description: Exotic plant species can evolve adaptations to environmental conditions in
 the exotic range. Furthermore, soil biota can foster exotic spread in the
 absence of negative soil pathogen-plant interactions or because of
 increased positive soil biota-plant feedbacks in the exotic range. Little
 is known, however, about the evolutionary dimension of plant-soil biota
 interactions when comparing native and introduced ranges. To assess the
 role of soil microbes for rapid evolution in plant invasion, we subjected
 Verbascum thapsus, a species native to Europe, to a reciprocal transplant
 experiment with soil and seed material originating from Germany (native)
 and New Zealand (exotic). Soil samples were treated with biocides to
 distinguish between effects of soil fungi and bacteria. Seedlings from
 each of five native and exotic populations were transplanted into soil
 biota communities originating from all populations and subjected to
 treatments of soil biota reduction: application of (i) fungicide, (ii)
 biocide, (iii) a combination of the two and (iv) control. For most of the
 investigated traits, native populations showed higher performance than
 exotic populations; there was no effect of soil biota origin. However,
 plants developed longer leaves and larger rosettes when treated with their
 respective home soil communities, indicating that native and exotic plant
 populations differed in their interaction with soil biota origin. The
 absence of fungi and bacteria resulted in a higher specific root length,
 suggesting that V. thapsus may compensate the absence of mutualistic
 microbes by increasing its root-soil surface contact. Synthesis.
 Introduced plants can evolve adaptations to soil biota in their new
 distribution range. This demonstrates the importance of biogeographic
 differences in plant-soil biota relationships and suggests that future
 studies addressing evolutionary divergence should account for differential
 effects of soil biota from the home and exotic range on native and exotic
 populations of successful plant invaders.
description: <strong>Sampling design
 </strong> Seed material and soil samples
 from five <i>V. thapsus</i> populations each were sampled in
 January and April 2014 in the exotic (New Zealand = NZ) and native
 (Germany) range, respectively. Populations were selected out of a pool of
 17 and eight populations (in NZ and Germany, respectively) with a minimum
 distance of 5km between populations. Populations from sites with extremely
 low or high soil pH were excluded in order to sample populations from
 comparable abiotic/edaphic site conditions. Populations chosen for the
 study experienced a mean annual temperature from 7.3-9.2°C (in Germany)
 and 8.7-10.3°C (in NZ), while having an annual average precipitation
 ranging from 485 to 708mm in Germany and 503 to 2208mm in New Zealand
 (Table 1). Seed material was kept dry at 4°C until the beginning of the
 experiment. Within each population, five topsoil samples were randomly
 taken in the upper 10cm of the mineral horizon and subsequently pooled by
 population to more encompass the heterogeneity of the site (Gundale et
 al., 2019). Soil samples were collected during the hemispheres’ respective
 vegetation period, i.e., in January 2014 in New Zealand and in May 2014 in
 Germany. Subsequent to sampling, all soils were transported in cool boxes
 and immediately stored at -80°C in the labs at Lincoln University (NZ) and
 Martin Luther University Halle (Germany), respectively. Samples from New
 Zealand were transported to Germany in April 2014 by maintaining the cold
 chain with dry ice and stored together with German samples at -80°C to
 stop microbial activity. After
 three (NZ) and six months (Germany), respectively, soil was gradually
 thawed. To reduce
 effects of differences in nutrient availability and soil structure, we
 used standard soil (see below) inoculated with soil washes from the
 different origins. Fresh soil from each of the ten populations was used to
 transfer soil-borne fungi and bacteria into a solution following Wagg et
 al. (2011). For this purpose, 100 ml of fresh soil were mixed with 500 ml
 deionised water and shaken by hand for ten minutes. Subsequently, the
 mixture was filtered through a 500 μm soil sieve. This procedure was run
 two times for each soil origin to increase the final amount of extracted
 solutions. The solutions obtained from this process were divided into four
 equal parts and subjected to treatments of Chlortetracyclin (80µg/l),
 Cycloheximid (80 µg/l), a combination of both (80 µg/l) or served as a
 control in order to reduce the total amount of bacteria (B), fungi (F),
 bacteria and fungi (BF) or none, respectively. To differentiate between
 possible direct physiological effects of the biocides and the true soil
 biota-mediated effect, the biocides were additionally applied to plants in
 sterilised soil without adding soil biota and compared to a control
 treatment with sterilised water. <strong>Greenhouse
 study</strong> The experiment took place in
 autumn 2014 in the greenhouse cabins of the Botanical Garden at Martin
 Luther University Halle. To prevent a contamination of samples and
 experimental units by ambient biota, we applied sterilisation procedures
 for the greenhouse cabins (0.3% Wofasteril E400, Kesla Pharma Wolfen GmbH,
 Germany), the seeds (rinsing 15min. 0.3% sodium hypochlorite solution) and
 the sand and soil used for seedling cultivation and growth (24 h at 180°C,
 8 h break, 24 h at 180°C). <i>Verbascum thapsus</i> seedlings
 grew on pure sand to facilitate later transplantation and were fertilised
 every 10 days with 0.4% Wuxal Universal (Hauert Manna Düngerwerke GmbH,
 Nürnberg, Germany). After ten weeks, plants were transplanted into pots
 with 2l of the sterilised soil/sand mixture. Pots were thereafter randomly
 assigned to biocide treatments and inoculated with the respective soil
 washes (10 ml microbial solution/1 l soil). Each ten plants of the ten
 origins were grown in all soil biota communities in a reciprocal design,
 i.e. originating from all populations and each subjected to all treatments
 of soil biota reduction, yielding a total number of 400 experimental units
 (i.e. individuals). Because of their fast growth during the experiment,
 plants were repositioned anew in two cabins after four
 weeks<b>.</b> Thereafter, all plants from population NZ2, NZ4,
 GE1, GE3 and GE5 were randomly allocated to cabin 1, and all other plants
 remaining from populations NZ1, NZ3, NZ5, GE2 and GE4 were newly arranged
 in cabin 2. Pots were randomly positioned on benches and saucers were used
 to avoid microbial cross-contamination from neighbouring pots. During the
 experimental period of twelve weeks, plants were watered every second day
 with deionized water, fertilised every 21 days with 0.4% Wuxal Universal
 and subjected to additional illumination from 5 am to 9 am and 5 pm to 9
 pm to ensure long-day exposure to light according to a 16h/8h day/night
 cycle. The temperatures ranged between 28°/15°C (day/night), accordingly.
 Every three weeks, rosette area size, leaf number and leaf length of the
 largest fully developed non-senescent leaf were monitored. For the
 calculation of leaf lifespan, leaves were marked after six and nine weeks
 to distinguish between old leaves that already existed at the previous
 monitoring date, the newly emerged leaves and the total leaf number at the
 initial date. By this, we were able to separate losses of previously
 present leaves from true increases because of emergences of new leaves
 (King 1994). Leaf lifespan was calculated by dividing the number of leaves
 per plant by the average of leaf production and leaf loss rates. After 12
 weeks, plants were monitored for survival and the largest fully developed
 non-senescent leaf was harvested to determine the leaf fresh weight, area,
 length, width and dry weight per individual (after 48h at 60°C). This
 information was used for the calculation of specific leaf area (SLA) and
 leaf dry matter content (LDMC). Total carbon and nitrogen on the leaf
 sample level were determined with a nitrogen analyser (vario EL cube,
 Elementar Analysensysteme GmbH) and used for C:N ratio calculation. Above-
 and belowground biomass was harvested separately. For the assessment of
 specific root length (SRL) roots were rinsed with water, and root area and
 length were measured suspended in water with a transmitted light scanner
 (Epson Expression 10000 XL, software package WinRHIZO). Both roots and
 shoots were dried for 72h at 80°C and weighed to obtain dry biomass by
 fraction and to calculate total biomass and root-shoot ratio
 (RSR).
description: <strong>country plant origin</strong>:
 <strong>PGE</strong>=Plant seed material from German
 populations; <strong>PNZ</strong>=Plant seed material from New
 Zealand populations <strong>country soil origin:
 SGE</strong>= Soil samples from German populations;
 <strong>SNZ</strong>= Soil samples from New Zealand
 populations <strong>RGR</strong>:
 calculated per week: RGR=lg(date of final harvest after 12 weeks)-lg(date
 of experimental start)/12 weeks
 <strong>monitoring measurement:</strong>
 <strong>0</strong>=experimental start,
 <strong>1</strong>=after 3 weeks;
 <strong>2</strong>=after 6 weeks;
 <strong>3</strong>=after 9 weeks;
 <strong>4</strong>=after 12 weeks