Germination and growth trials were undertaken from January – October 2018. Experiments were undertaken in incubators and growth cabinets which could be maintained at temperatures reflective of the altitudes that these species currently experience in Australia. Incubators were able to maintain low temperatures experienced in winter and had a PAR of 21 µmol m−2 s−1 which is similar to that experienced under snow. Growth cabinets had a PAR of 485 µmol m−2 s−1 which equated to that experienced on a cloudy day (510 µmol m−2 s−1).
Temperatures were set each month using average weather station data from around 1000 m (Hill Prison Camp 1006 m) or 1700 m asl (Perisher Valley AWS 1738 m) for each month. To calculate a typical day time temperature for any month, we identified the maximum temperature for each day within a month then took the midpoint between the highest and lowest temperature. We followed the same method to identify the night time temperature using minimum temperatures for each night of a month. Day and night length throughout all experiments reflected, and were adjusted to, the months we simulated.
Germination
Germination of H. aurantiacum used seeds from three sites collected over the 2017/2018 summer (termed New) and older seeds from four sites (termed Old) that had been stored in paper bags at room temperature for up to four years. For each site, eight replicates of each of three temperature treatments were set up; temperatures replicating 1000 m, 1700 m, and 200 m asl. Each replicate consisted of 25 seeds placed in a petri dish with filter paper moistened with distilled water. In all, there were 144 petri dishes containing 3600 seeds of H. aurantiacum. Petri dishes in temperatures mimicking alpine temperatures were placed in incubators, while the H. aurantiacum trial at 200 m was placed in the glasshouse.
Due to the eradication program and resultant scarcity of H. pilosella only one site with new seeds collected over the recent summer and three sites with old seed collected in the last 5 years were available. These sites are within metres as there is only one invasion area near Charlotte Pass. As seed numbers were low we could only measure germination at temperatures mimicking 1700 m. The new seed site had only enough seeds for 7 petri dishes while the other sites had 8 petri dishes each. Thus, the H. pilosella seed trial contained 31 petri dishes and 775 seeds.
Petri dishes were checked for germination every two weeks and the experiment was terminated on 10th July after just over 15 weeks. Tetrazolium testing of viability of the remainder of seeds in the petri dishes was attempted but without success as the hawkweed seeds are extremely small and seeds did not show any stain even when attempted on new seed material.
We use the term seedlings to be small plants grown from seed in this experiment. To avoid confusion in this study, we use the term ‘ramet’ as any attached plantlet that has grown on a stolon from its parent seedling or plantlet (See Growth of Seedlings below), while ramets that have been separated from stolons and are independent are identified as ‘plantlets’ (see Growth of Plantlets below).
Growth of seedlings
As seeds germinated they were planted under the same conditions under which they were germinated, in a seedling plug before being transferred to small pots containing a mixture of general potting mix and coarse river sand (2:1). At the beginning of the seedling growth trial, 40 H. aurantiacum seedlings at the three-leaf-stage were potted up from seedling plug trays into small pots (130 mm diameter). From the 1000 m germination trial, 20 were transferred into a growth cabinet set to 1000 m conditions and another 20 from the 1700 m germination trial were transferred into a growth cabinet set to temperatures at 1700 m (April temperatures). Twenty H. pilosella seedlings were also transferred to the cabinet set at temperatures reflecting those at 1700 m altitude. The seedlings were watered twice a week and fertilized once a month with Osmocote Plus Organics all purpose liquid fertilizer (N: 15.4%, P: 0.0%, K: 6.0%). Plants were moved back into the incubators in June in order to experience winter temperatures.
After 188–189 days, plants were harvested. Stolons and leaves were separated from root, and new ramets were counted and separated. All plant parts were dried at 70 °C for 10 days and then weighed to give dry biomass.
Growth of plantlets
Two large pots of H. aurantiacum and one of H. pilosella were provided by the Department of Primary Industry. These pots contained multiple plants each with large numbers of stolons with numerous ramets attached. H. pilosella plants were at least 3 years old and grown from vegetative stock so three years represents a minimum age. H. aurantiacum pots were probably around two years old. Between 27th February and 1st March, ramets were separated and potted up into 130 mm diameter pots. In all, 45 H. aurantiacum and 33 H. pilosella ramets were separated into mother plant (those that had stolons emanating from them) and ramets (those who were found at the end of stolons emanating from a mother plant) (Fig. 1). Only ramets of at least 4 to 5 leaves were used; the rest discarded. Initially for H. pilosella there were 4 mother plants with an average of 4.75 ± 1.50 (s.d) stolons. There were 5 H. aurantiacum mothers with an average of 5.83 ± 9.02 (s.d) stolons. Only two of the mother plants from H. pilosella and one from H. aurantiacum grew and had leaves.
Experimental design for measuring growth of adult plants.
With these established plantlets, there was only room to grow each species under one altitude that best reflected its invasion conditions. Initially, H. aurantiacum plantlets were placed into a Thermoline growth cabinet set to temperatures reflective of 1000 m asl. H. pilosella plantlets were placed into a separate Thermoline growth cabinet set to temperatures reflective of 1700 m asl. At the beginning of winter due to limited space, we placed a random set of 12 pots of each species into incubators after repotting in 30 cm pots, to ensure winter temperatures were maintained and harvested all other plants (day 69 for H. aurantiacum and day 126 for H. pilosella, Fig. 1). Harvested plantlets were separated into above and below ground biomass, and individual ramets before drying and weighing.
On day 148 (26th July 2018) H. aurantiacum plantlets were harvested as the 12 big plants had completely filled the pots. We continued to monitor for one week past our decision as they experienced some very high temperatures for 2 days at the end of our sampling when the cabinets failed to maintain set temperatures. Daytime temperatures reached 38–40 °C for a few hours on these days. Rather than harvest immediately, we harvested after a week (day 148) to observe any deaths following this event. Above and below ground dried biomass and individual ramet dry weight were recorded. The remaining 12 H. pilosella plantlets also received warmer than planned heating during July as well, but only increased to 24 °C which was not considered extreme for a warm day at this altitude. They were harvested on day 219 (9–11 October 2018) and above and below ground biomass as well as individual ramets were dried and dry weight recorded.
Analysis
For H. aurantiacum the proportion of seeds germinated per petri dish was compared using a restricted maximum likelihood model with age and altitude as fixed factors and site nested within age. For H. pilosella, sites were compared as a fixed factor using a standard least squares model as there were no replicate sites nested within age and a lack of seeds meant we could only compare sites at the 1700 m altitude.
We compared the number of leaves, number of stolons and dry biomass measurements [above ground biomass, below ground biomass, total biomass, proportional mass in ramets and the root:shoot ratio] using mixed models with a binomial distribution and log link function. Stolon weight and leaves were included as above ground biomass while total weight included leaves, stolons, ramets and roots. Where significant effects were found, Tukeys HSD tests were used to identify where differences lay.
Source: Ecology - nature.com
