Study site and transplantation experiments
In low-flowering years few or no tillers flower in the field, and even in high-flowering years only a minority (<20%) of tillers on each plant flower (Rees et al., 2002). In order to improve our chances of having both flowering and vegetative samples, we took leaf samples from unmanipulated plants in the field, and from manipulated plants that were transplanted to lower-elevation (warmer) or higher-elevation (colder) sites to make flowering respectively more or less likely (Table 1). In 2016, unmanipulated Chionochloa pallens plants at the main Mt Hutt site at 1070 m elevation (43⁰32ʹ S, 171⁰ 33ʹ E) were selected with leaf samples taken from three marked tillers on each of 10 marked plants, with another 30 tillers selected and sampled the following two years. None of the tagged plants at the control plot flowered in 2017 and 2018, but they did in 2019.
Several transplantation experiments to higher or lower altitudes were carried out to manipulate flowering in C. pallens . Each group of transplanted tussocks was identified by the year of transplantation and the treatment provided. Ten separate plants from the vicinity of the control plot were moved to near sea-level at the University of Canterbury (UC, Christchurch; 43° 31ʹ S, 172° 35ʹ E, 15 m a.s.l.) for the inductive summer period in December 2016 to March 2017 (17Hot transplants) and then transplanted back to the 1070 m site. Leaf samples from tagged tillers (both transplants and control plants) were collected at different time points through the year (January 2017, March 2017, October 2017 and January 2018; between 11:00 am and 2:00 pm) and were used for the gene expression studies. Leaf samples collected during the inductive summer period (January 2017) were also used for the transcriptomic analysis as described below. Two other sets of 10 plants each were permanently transplanted in December 2015 from the control site at 1070 m to different altitudes: to UC (16Hot) and to 1520 m on Mt Hutt (16Cool). Leaf samples were collected at different seasonal time-points (between 11:00 am and 2:00 pm) including summer (January 2016), autumn (March 2016), spring (October 2016) and autumn (post flowering; March 2017).
In all the experiments, all leaf samples were collected and put directly into dry ice within 20 seconds and stored at -80 ºC until further analysis. The tagged tillers were labelled and subsequent behaviour (flowering or not) was recorded in the following year. The stored leaf samples could then be identified as being from tillers that subsequently flowered or remained vegetative, and suitable samples were selected for downstream analysis.