Differences in vegetative growth of two invasive hawkweeds at temperatures simulating invaded habitats at two altitudes

Hieracium pilosella and H. aurantiacum are invading alpine regions in New South Wales, Australia. In a glasshouse experiment we investigated germination and growth rates of these two species at temperatures simulating the altitudes where invasions are occurring from autumn to spring. We measured germination rates, growth rates and the development of stolons and ramets using seedlings and plantlets from established plants. Germination was low in H. aurantiacum and unaffected by altitude or seed age. H. pilosella showed site to site variability in germination but had greater germination. No species produced flower spikes. Both species grew rapidly and put at least twice as much biomass into roots compared to shoots. H. aurantiacum could begin to produce stolons after 27 days and seedlings grew a little larger than for H. pilosella. Hieracium aurantiacum put significantly more resources into ramets, allocating between 4–15% of biomass. H. pilosella produced 2.6 stolons month−1, in contrast to 9.8 stolons month−1 for H. aurantiacum. Furthermore, plantlets from established plants had vastly different growth rates. Plantlets of H. aurantiacum produced 2.1 leaves day−1 from late summer to winter where H. pilosella was 3 times slower for the same period but faster following winter. Both species were able to maintain strong growth over cooler months suggesting hawkweeds have the capacity for fast growth in the invaded range under high nutrients and lower competition. H. aurantiacum is likely to be a more effective invader than H. pilosella spreading through stolons and the development of weed mats.


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 10 th 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 27 th February and 1 st 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.
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.
www.nature.com/scientificreports www.nature.com/scientificreports/ On day 148 (26 th 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.   Fig. 2). There was no difference in germination rate between old and new seeds (F 1,4.914 = 0.571, P = 0.485) nor an interaction between the two main effects (F 1,9.30 = 0.771, P = 0.490). Sites were highly variable with the proportion germinating ranging from 0.04 ± 0.01 to 0.31 ± 0.04 suggesting significant site to site variability in viable seed production. A greater proportion of seeds germinated for H. pilosella, with similarly high levels of variation in the proportion of seed germinating from different sites, but with no obvious relationship with age of seeds (F 3,27 = 13.100, P < 0.0001, Fig. 3). Seeds of H. aurantiacum began germinating after 5 days reaching a maximum after a month, while seeds of H. pilosella began germinating after about 2 weeks and reached maximum after 5 weeks.

Discussion
Orange and mouse-ear hawkweed grew very quickly under the conditions provided in this experiment. Both species put considerably more into below-ground growth relative to above ground, even when ramet production was included. Below-ground capacity is very important in resource-poor habitats [22][23][24] and strong below ground growth may increase resilience during snow covered winter periods, although no losses in leaves occurred at below freezing temperatures. Both species are likely to rely heavily, in the field, on the development of below ground biomass to aid competition and survival. Weigelt et al. 25 found that competition occurred largely below ground in H. pilosella although it was less competitive under water limitation.
Invasive species are considered to be advantaged by developing strong growth in invaded ranges 16,17 although field measurements are rare 26,27 . Invading plants would be expected to develop improved competitive attributes (EICA hypothesis 28 ), however invaded range features may influence this. Rates were much quicker in our experiments than those measured in the field, perhaps associated with the well watered and non competitive conditions in this experiment. Thus, they more closely represent intrinsic growth rates. In the field in New Zealand, root:shoot ratios for H. pilosella 29 and overall growth rates were much lower 25 , suggesting competition is an important modifier. Certainly H. pilsosella tended to colonise poor quality and degraded soils in New Zealand 30 and in native regions 31 which reflects the conditions in the invaded area in Australia. It is known to influence resource acquisition in native species 2 . Together with our results, this suggests that hawkweeds have the capacity for fast growth in the invaded range under conditions of higher nutrients and lower competition.
We found a large difference between the two species in the allocation of resources to vegetative reproduction which were only partially explained by the different altitudes in which the two species have invaded in Australia. H. aurantiacum put significantly more resources into ramets, allocating up to between 4-15% of biomass depending on age. Stolon production began when seedlings were around 4 weeks at termperatures mimicking 1000 m and at 6 weeks at 1700 m indicating a strong vegetative growth response and was a significant component of biomass. In contrast, H. pilosella seedlings did not produce stolons with ramets and plantlets only produced stolons when plants were over 1 year old and probably closer to two years old. The rate of production of stolons was slow (2.6 stolons per month), in contrast to 9.8 stolons per month for H. aurantiacum. However, rates of stolon production in H. pilosella was about twice that recorded further south in Victoria (~5.5 stolons per month 32 ) and greater than field plantlets in New Zealand (1.6 stolons per plantelet 33,34 ). In New Zealand mouse-ear and king www.nature.com/scientificreports www.nature.com/scientificreports/ devil HW relied almost exclusively on clonal growth for population growth but clearly new incursions were a result of seed dispersal 29 . Relative to king devil HW, H. pilosella produced less seed, but more daughters which established further from the parent 29 . Our results suggest that H. aurantiacum is even more effective in spreading from vegetative growth. Furthermore, given that neither species produced flower spikes during our experiments, it suggests that H. aurantiacum was a more effective invader than mouse-ear in the NSW alps, spreading through stolons and the development of weed mats.
Both species were able to maintain strong growth, except when temperatures reflected winter norms, particularly at temperatures mimicking the higher altitude, when both species did not produce new leaves until temperatures came above zero. After the first five months of growth, seedlings had produced leaves at a rate of about 0.5 leaves day −1 . Older plantlets grew more quickly; plantlets of H. aurantiacum producing 2.1 leaves day −1 from late summer -winter temperature settings where H. pilosella was 3 times slower (0.7 leaves day −1 ) for the same period but it dramatically increased production when temperatures became wamer, and overall produced 1.4 leaves day −1 . The increase in growth rate of established plants, suggests that, in order to achieve eradication, there is a window of opportunity of about 18 months prior to flowering and before a rapid increase in growth rate when invasion patches can be eradicated before creating significant breaks in native plant cover.
Hieracium pilosella had seeds with better germinability than H. aurantiacum, although neither were as high as measured by Bear et al. 32 who found up to 89% germination for 3 month old, dry stored seeds. Better germination may be important if H. pilosella has greater allocation to reproduction rather than vegetative spread 18 . Hieracium pilosella benefits more from increased sexual reproduction in its invaded range than in native range 18 . Beckman et al. 18 only found 7% germination for H. pilosella in Germany. In NZ the high density of seed fall produced by H. pilosella, coupled with high seedling survival was thought to be important in the invasion of this species 29,33 . Seeds were equally viable with age, indicating a longer term seed bank may be able to develop. Further research on longevity in the field would be beneficial.   www.nature.com/scientificreports www.nature.com/scientificreports/ Hieracium aurantiacum may well be able to germinate at lower altitudes but hot conditions experienced over the summer in these more coastal areas, may well limit the probablity of survival. About 10% of H. aurantiacum seeds germinated at temperatures mimicking low altitudes, less than at lower temperatures mimicking higher altitudes, but seedlings did not survive the hot conditions experienced during the experiment. Despite this susceptibility, a cooler summer may allow establishment of seedlings and it is clear from this experiment that growth will be rapid. Once established, plants of both species appear to be resilient to hotter conditions. The hotter conditions experienced during the failure of the growth cabinets at day 146 (up to 24 °C) for H. pilosella, and the very hot conditions (>35 °C) for H. aurantiacum at 1000 m had no visible effect on either species.
This study has shown the enormous capacity for growth of these two species of invading hawkweeds in the alpine regions in NSW, Australia. Vegetative reproduction is critical in the early stages of invasion to occupy space. H. aurantiacum was far more effective in vegetative growth compared to H. pilosella, suggesting its rate of spread will be much greater than H. pilosella. Both species, once established, appear to have significant mechanisms for tolerance derived from a large below ground biomass but also an apparent ability to easily cope with heat events when adequately watered. Further work is needed to understand hawkweed responses to interactions between temperature and other variations in climate that vary with altitude (rain/snowfall, wind etc). Understanding the negative effects of competition and nutrient limitation in the field in these species will greatly enhance our capacity to model spread in these species.