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.