1. 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.
2. 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.
3. 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.
4. 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.

Sampling design

Seed material and soil samples from five V. thapsus 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.

Greenhouse study

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). Verbascum thapsus 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. 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).

country plant origin: PGE=Plant seed material from German populations; PNZ=Plant seed material from New Zealand populations

country soil origin: SGE= Soil samples from German populations; SNZ= Soil samples from New Zealand populations

RGR: calculated per week: RGR=lg(date of final harvest after 12 weeks)-lg(date of experimental start)/12 weeks

monitoring measurement: 0=experimental start, 1=after 3 weeks; 2=after 6 weeks; 3=after 9 weeks; 4=after 12 weeks