Soil sampling and greenhouse experiments
Soil samples were collected in the early autumn after the ending of the
growing season and seed dispersion. Thus, the SSB contained transient
and persistent seeds. After a field survey, fifteen sampling sites were
randomly selected, each site containing the three species of shrubs
together with a herbaceous control area (outside the shrub canopies) in
close proximity to each other (Fig. 2). The distance between any two
sampling sites was at least 100 m to exclude spatial autocorrelation. In
each sampling site, beneath each shrub individual (patch), after
removing coarse litter (> 2 cm) 10 soil cores (subsamples)
were randomly collected, to a depth of 10 cm, with a 5 cm diameter auger
and then the subsamples were pooled for each patch (totally 60 soil
samples were collected). Then, the soil samples were stored at 4 ºC to 5
ºC for cold stratification (Dreber, 2011) for a period of 25 days.
Subsequently, each soil sample was distributed evenly over a mix of
sterilised potting soil and sand in the trays of 25 cm × 35 cm (60 trays
in total). The sterilised soil layer was 3 cm and the field collected
soil layer was maximum 2 cm in the thickness in the trays. The
germination trays were labelled and distributed randomly on benches in
the greenhouse with natural light and temperature conditions (varied
between 15 °C and 26 °C ) and irrigated every second day (Niknam et al.,
2018). In addition, six control trays containing only sterile material
were randomly placed between the sample trays to test for seed
contamination.
Identifiable germinated seedlings were counted and removed from the
trays every week. The seedlings were identified to species level.
Seedlings that could not be identified were transplanted to pots to
allow further growth until identification was possible.
After a period of seven months, no further seedlings were emerged.
Therefore, the trays were left to dry for two weeks and then the samples
were reirrigated for another one month to help seed dormancy breaking.