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.