Soil sampling and greenhouse experiments
Soil sampling was carried out in the early autumn after the end of the
growing and seeding season. Thus, the germinable seeds contained
transient and persistent components of the SSB. After a field survey, we
randomly selected 15 replicate sampling areas, each containing the three
shrub species and a herbaceous patch (control) in close proximity to
each other (Fig. 2). The distance between any two sampling areas was at
least 100 m to exclude spatial autocorrelation. In each sampling area,
under each shrub, after removing coarse litter (> 2 cm) 10
soil cores (subsamples) were collected at random, 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). The soil samples were
transported to the laboratory and were stored at ca. 4 ºC for cold
stratification for a period of one month (Miller and Cummins, 2003).
Subsequently, soil samples were transported into the greenhouse and each
sample was spread over a mix of (1:1) sterilised potting soil and sand
of a thickness of 3 cm in free draining plastic trays of 25 cm × 35 cm
(totally 60 trays). Six control trays containing only sterile potting
soil and sand, were placed at random with the samples to test for
contamination of samples from greenhouse or nearby seed sources. All
trays were kept under natural light and temperature conditions and were
watered to keep them moist (Niknam et al., 2018). Air temperature varied
between 14 °C and 25 °C. Germinated seedlings were identified, counted
and removed once they reached an identifiable stage. Seedlings that
could not be identified immediately were transplanted to pots to allow
further growth until identification was possible. No seeds were
germinated in the control trays.
After a germination period of seven months, no further seedlings were
observed. Therefore, the trays were left to dry for two weeks and then
the samples were rewetted and kept for another one month to encourage
seed dormancy breaking.