Seed collection, growth conditions and experimental setups
We collected seeds from Taraxacum officinale populations in
places where the species is native (Europe) or introduced (South
America). Populations in the native range were París, Lyon and Marsella
(France) while those of the introduced range were Antofagasta (23° 37´
S), Caldera (27° 06´ S), La Serena (29° 54´ S), Viña del Mar (33° 01´
S), Talca (35° 27´ S), Concepción (36° 57´ S), Valdivia (39° 80´ S),
Coyhaique (45° 61´ S) and Punta Arenas (53° 10´ S). Four to five seeds
were collected per individual (N = 60 maternal plants) and pooled by
population. For each population, 200 seeds were germinated on wet paper
in Petri dishes at room conditions (24 ± 4 ºC). Those plants were grown
in individual pots (3 L) filled with commercial soil in a greenhouse and
watered on demand. This first generation (F1) of plants
from each population were grown until they produced seeds, which were
used to obtain the experimental F2 plants.
Besides the collection of seeds from each population, we also collected
rhizospheric soil at each site. We dig the soil around the roots of 50T. officinale plants and carefully transferred it to a common bag
for each site. For the experiments, T. officinale plants were
randomly assigned to three treatments: “native soil microbiome
(M+)”, “removed soil microbiome
(M-)”, and “re-inoculated soil microbiome
(Mr)”. The M+ treatment consisted
in groups of 500 cc plastic pots (one seedling per pot) filled with a
mix of native rhizospheric soil and sand in a 2:1 proportion. For the
M− treatment, pots were filled with the same native
rhizospheric soil but previously sterilized by autoclaving (121 °C, 90
min.). Lastly, pots in the Mr treatment were initially
the same as those in M− treatment that were later
irrigated with 100 mL of a solution containing the native soil
microorganisms (i.e., 200 gr of native soil diluted in 1,0 L of
tap-water). Autoclaving eliminated soil microorganisms, allowing us to
study the effect of local microbiomes on the fitness-related traits inT. officinale plants. Since autoclaving can also alter the
availability of soil nutrients (de Deyn et al. 2004), we evaluated the
content of nitrogen (N) and phosphorus (P) in autoclaved and
non-autoclaved soils (n = 12 each). No differences were found in
the availability of the two nutrients (t = 0.71; p = 0.26 andt = 0.97; p = 0.41).
One week after appearance of the first true leaf, the seedlings in the
plastic pots were transferred to a glasshouse under semi-controlled
conditions (air temperature: 26 ± 3ºC; relative humidity: 75 ± 3%; and
solar radiation: 1160 ± 80 µmol
m-2s-1). Each pot was irrigated with
75 mL of tap-water (or tap-water with diluted rhizospheric soil in
Mr) every two days. In each experiment, the pot
positions were weekly randomized within the glasshouse. Inter-pot
distance was sufficient to prevent mutual shading influence. The two
experiments lasted 100 days and in both, plant survival as well as
flower production were recorded every 5 days. At the end of the
experiment, we recorded total dry biomass (including fallen leaves) of
plants. The plant material was oven-dried at 70 °C for 72 h.
In experiment 1, we studied the effect of local and introduced soil
microbiome on the performance of T. officinale plants from
populations of both the native (Europe) and the introduced (South
America) range. This experiment was carried out simultaneously in Europe
(52º 12´N; 00º 04´E, Cambridge, UK) and in South America (35º 24´S; 71º
37´W, Talca, Chile). Greenhouse conditions in the UK were similar to
those in Talca. We used F2 seeds from Paris, Lyon, and
Marseille to represent the native range, while those from La Serena,
Talca, and Punta Arenas were selected to represent the introduced range.
In each location, a total of 225 seedlings per population were
established where 75 was the number of plants randomly assigned to any
of the soil microbiome treatments [control (M+),
sterilized (M−) or sterilized and reinoculated
(Mr)]. To compare the effect of the microbiome on
the fitness-related traits of T. officinale between native and
introduced range,
In experiment 2, we studied the effect of the soil microbiome on the
performance of T. officinale plants from populations collected
along a latitudinal gradient in the introduced range (Antofagasta,
Caldera, La Serena, Viña del Mar, Talca, Concepción, Valdivia, Coyhaique
and Punta Arenas) (Suppl. Table 1; Suppl. Figure 1). This experiment was
carried out in Talca, Chile. A total of 90 seedlings per population were
established where 30 were randomly assigned to any of the soil
microbiome treatments [control (M+), sterilized
(M−) or sterilized and reinoculated
(Mr)]. Apart from the common procedures indicated
above, in this experiment the individual performance variables were
integrated through the calculation of a “Performance Index”. For each
plant, we summed the scaled values (i.e. , between 0 and 1) of
their three recorded response variables (survival, biomass and flower
number), after being weighted by their proportional contribution to the
fitness. The proposed weighting values correspond to 0.5 for survival,
0.3 for flower production, and 0.2 for biomass. For example, a
performance index of 1 corresponds to a plant with the highest
performance in all variables, while lower index values indicate lower
performance. It is worth mentioning that in the case of survival, each
individual value corresponds to the proportion of survivors within their
respective experimental group at the end of the experiment.