2.2. Materials and Methods 2.2.2. Seed germination experiments Seeds used in the germination and predation experiments were collected in the Canberra region of south-eastern Australia during December and January of 1990/91 and 1991/92. They were stored in paper bags at room temperature (approximately 22°C) until used, no later that 10 months after the collection date. Germination experiments were carried out in growth cabinets, a glasshouse and at various field sites (Table 2.1). 2.2.2.1. Growth cabinet experiments Dormancy and the effect of temperature on germination of H. gramineum seeds were assessed in the following tests. These trials were supplemented by examining the germinability of seeds from H. gramineum infested with A. hyperici. Unless stated otherwise, all petri dishes (replicates) contained 0.005 g of seed (300.5 seeds ± 4.8 s.e., n = 15) scattered over a Watmans No. 1 filter paper, which had been moistened previously with 6 mL of distilled water (or gibberellic acid - see below). Dishes were then sealed and incubated in an artificially lit growth cabinet with a 30°C day (8 hours, with a photon flux density of 7 0 - 1 1 0 pmol n r2 s_1) and a 20°C night (16 hours) until completion of the experiment, 20 days later. Table 2.1 Location of study sites used in various experiments investigating aspects of the seed ecology of H. gramineum. Experiment Habitat Site Site latitude & longitude Field germination Grassland Border East, ACT Border West, ACT Horse park, ACT Radio CY, ACT Smith’s Paddock Gl, ACT Smith's Paddock G2, ACT 35° 10' S, 149° 09' E 35° 10’ S, 149° 0 9 'E 35° 10' S, 149° 08' E 35° 13'S, 149° 0 2 'E 35° 17’ S, 149° 05’ E 35° 17 S, 149° 05’E Woodland Border North, ACT Border South, ACT Honeysuckle Creek, ACT Kowen Forest, NSW Smith's Paddock W l, ACT Smith's Plantation, ACT 35° 10’ S, 149° 09’ E 35° 10’ S, 149° 09’ E 35° 35’ S, 149° 00’ E 35° 16’ S, 149° 15'E 35° 17 S, 149° 0 5 'E 35° 17 S, 149° 0 5 'E Viability after field-exposure Grassland Beechworth Gl, Victoria Beechworth G2, Victoria Mt. Ainslie Gl, ACT Mt. Ainslie G2, ACT Smith's Paddock G 1, ACT Smith's Paddock G2, ACT 36° 2 3 'S, 146° 4 4 'E 36° 23' S, 146° 44' E 35° 17'S, 149° 10’E 35° 17 S, 149° 10’E As above As above Woodland Beechworth W l, Victoria Beechworth W2, Victoria Mt. Ainslie W l, ACT Mt. Ainslie W2, ACT Smith's Paddock W l, ACT Smith's Paddock W2, ACT 36° 2 3 'S, 146° 43’E 36° 2 3 'S, 146° 4 3 'E 35° 17 S, 149° 10' E 35° 17’ S, 149° 10’E As above 35° 17 S, 149° 05’E Seed bank N/A Beechworth 1, Victoria Beechworth 2, Victoria Farrer Ridge 1, ACT Farrer Ridge 2, ACT Mt. Taylor, ACT Smith's Paddock, ACT 36° 23’ S, 146° 44’E 36° 23’ S, 146° 43’ E 35° 23’ S, 149° 05’ E 35° 23’ S, 149° 05’ E 35° 23’ S, 149° 04’ E As above Seed predation Grassland Smith's Paddock Gl Mt. Taylor Farrer Ridge 1 Farrer Ridge 2 As above As above As above As above Woodland Smith's Paddock W 1 Smith's Plantation Farrer Ridge W Mt. Ainslie Wl As above As above 35° 23’ S, 149° 05’E As above Chapter 2 - H. gramineum reproductive ecology 25 (a) Dormancy The existence of dormancy in H. gramineum seed, and the means by which any such dormancy could be broken, was tested by subjecting seeds to five treatments during, and/or prior to germination: (1) After-ripening - seeds were germinated at 4-weekly intervals, from the date of collection, for 24 weeks. Subsequently, germinations were conducted after 40, 55 and 77 weeks. (2) Gibberellic acid - seeds were germinated on filter paper moistened with a 500 ppm solution of gibberellic acid (GA3). (3) Cold stratification - seeds were placed on moistened filter paper and stored at 4°C in the dark for 3 weeks. (4) High temperature - seeds were stored between 40° and 50°C in the dark for 3 weeks. (5) Washing - seeds were washed for 24 hours in running tap water. (b) Temperature and darkness Germination trials were conducted using 6-month old seeds at alternating day/night temperature regimes of 15/5, 20/10, 25/15, 30/20 and 35/25°C. Petri dishes used in the dark treatments were randomly interspersed amongst light treatments of a given temperature regime. They therefore received the same temperature alternations but were wrapped in aluminium foil for the duration of the experiment. (c) Herbivory Seeds from plants subjected to herbivory by A. hyperici in a glasshouse were compared with seeds from plants simultaneously propagated under identical conditions, but kept free of infestation by A. hyperici. Seeds were randomly selected from those produced by five mite-infested plants (+mites) and two mite- free plants (-mites). Each of 15 petri dishes for each seed source contained 20 seeds and were treated with a 500 ppm solution of gibberellic acid, as above. 2.22.2. Field experiments (a) Field germination experiment Seeds of H. gramineum were sown into rings of PVC piping, 12 cm diameter x 3 cm high and pushed into the soil of field sites leaving a 1 cm high rim protruding above the soil surface, thereby shielding the seeds from wind and rainwash. The experiment had a factorial design with six treatments (2 sowing times x 2 habitats x 2 micro-environments) applied to two species, as follows: (1) Sowing season - two seasons: seeds sown in autumn (late May), or in spring (early October) 1992. Seeds for the spring germinations were sown adjacent to those sown in autumn. (2) Habitat - two habitats: native grassland (dominated by Themeda triandra) or woodland (dominated by various combinations of Eucalyptus blakelyi, E. macrorhyncha, E. melliodora, E. nortonii and E. polyanthemos, with a grass understorey comprising T. triandra, Poa sieberiana, Danthonia spp. and native forbs). (3) Micro-environment - seeds were sown onto undisturbed, usually bare soil in two micro-environments: either sheltered on the southern (shady) side of a large grass tussock, or in an inter-tussock gap of 15 - 30 cm diameter. (4) Seeds - seed batches of two types were sown: (i) 0.005 g of pure H. gramineum seeds, and (ii) 0.005 g of H. gramineum seeds mixed with 25 H. perforatum seeds. Seed-free controls (for chance germinations within plots) confirmed that negligible germinations occurred unless seeds were sown into the plots. Six grassland and six woodland field sites were used in the experiment, each separated from one another by at least 500 m. All but one site were within the Australian Capital Territory (Table 2.1). The total number of germinations, including those of seedlings that subsequently died, was scored every four - six Chapter 2 - H. gramineum reproductive ecology 27 weeks for the nine months of the experiment, commencing two weeks after the 'autumn sowing'. (b) Measurement of light and soil temperature regimes Summer surface soil temperature (°C) and light intensity (photosynthetically active radiation, pmol n r 2s_1) were measured adjacent to grass tussocks and in inter-tussock gaps, in woodland (Eucalyptus spp.) and grassland (Themeda triandra) habitats representative of field sites used in the field germination experiment. Measured on a sunny day in mid-summer (December) 1993, these measurements quantified the likely temperature and light regimes experienced by H. gramineum seeds in the two environments. Twenty-six measurements were taken in each: thirteen adjacent to grass tussocks and thirteen in inter-tussock gaps. The data were compared by 2-way analysis of variance using Statview-4 (Abacus Concepts, Statview 1992), after logarithmically transforming the data. Un-transformed arithmetic means are presented in the results. (c) Seed viability after field-exposure H. gramineum seeds (0.005 g) were enclosed in fine mesh (< 0.25 x 0.25 mm holes) nylon bags, randomly assigned to a treatment within a factorial design and pegged to the soil surface, though many became buried by litter with time. The experiment comprised 11 treatments (3 field sites x 4 time periods x 2 habitats x 2 micro-environments), as follows: (1) Field region - three regions: Beechworth (Victoria), Mt. Ainslie and Black Mountain (ACT; see Table 2.1 for locations). (2) Time - seed bags were retrieved from the field after 3, 6, 12 and 18 months. (3) Habitat - two habitats (grassland and woodland), each twice replicated at each site. (4) Micro-environment - two micro-environments: either sheltered on the southern side of a large grass tussock, or in an inter-tussock gap. After collecting seed-bags from the field, their contents were germinated in vitro, using the same techniques employed in the growth cabinet experiments. Comparisons of the number of germinations were made between treatments. For each time period, the mean number of germinations was also compared with laboratory-stored seed of the same age, though this comparison was not included in the statistical analyses. 2.2.23. Glasshouse experiments (a) Seed bank survey (1) Survey Design: This factorial design survey comprised two soil depths, at locations beneath or distant to a plant, replicated four times at each of six field sites (see Table 2.1). The samples were taken using a 125 mm x 150 mm quadrat. One location was sampled immediately beneath a reproductively mature H. gramineum individual and the other, one metre away from the same and surrounding conspecifics, in an arbitrarily selected direction. Two soil depths, 0 - 1 cm and 1 - 3 cm, at each location within the site were sampled. Paired locations were separated from other replicates by 6 - 10 m. (2) Procedure: Samples were oven-dried for 5 days at 40° - 45°C before recording the mass of each sample. They were then evenly spread within 13 x 7 x 4.5 cm trays and arranged in a fully randomised experimental design within a glasshouse, and watered daily with tap water. After 30 days, the number of H. gramineum seedlings germinating from each sample was recorded. All seedlings were then removed and the soil turned and watered for a further 25 days to check for further germinations. The experiment was terminated after this second period, since few germinations occurred. The total number of germinations from both periods in each soil sample was combined and used in analyses. (b) Effect of seed burial on germination Germination of H. gramineum seeds sown at four soil depths (3, 1, 0.5 and 0 cm) was compared. Seeds (0.005 g) were evenly spread onto potting soil within 9 cm x 9 cm square pots of 8.5 cm depth. In all but the surface treatments, in which seeds were sown onto the soil surface, seeds were then covered with soil to the Chapter 2 - H. gramineum reproductive ecology 29 appropriate depth, ensuring that the soil surface was consistently 1.5 cm below the rim of the pots. Pots were placed under a misting spray within a glasshouse at 20 - 30°C, having randomly assigned one pot from each soil depth to each of 12 blocks within a randomised block design. After 40 days, the number of germinations in each pot was scored. In document The ecology of Hypericum gramineum with reference to biological control of H. perforatum by the mite, Aculus hyperici (Page 39-45)
