2.2 Leaf Movement Assays
To determine circadian period in natural populations of B .stricta , we adapted the protocol from Tracking Rhythms in Plants
(TRiP, Greenham et al. 2015). For each of the 30 populations, 10 to 20
family lines were selected for circadian analysis, and within each
family, 18 replicates were included. We planted seeds into Redi-Earth
potting mix (Sungro, Agawam, MA, USA) in 1-cm diameter pots and placed
the pots into growth chambers (Percival Scientific, Perry, IN, USA) for
germination using a 12-h/12-h light/dark cycle and 21 °C/18 °C day/night
temperature cycles. After six days, seedlings were moved to 14-h/8-h
light/dark cycle for entrainment; temperature conditions during
entrainment simulated early May conditions in Laramie, WY with hourly
cycling for temperature from 4 °C to 17.5 °C (Table S1). After seven
days in the entrainment conditions, seedlings were transferred to
imaging stands and placed in constant light and temperature (17.5 °C)
conditions for 24 h prior to imaging. Images were taken every 15 min for
five days to ascertain leaf positions, and stacked images of leaf
movements were analyzed in MatLab (The MathWorks, Inc.) through the TRiP
pipeline to determine circadian period (i.e., the duration of one
endogenous cycle). These germination and entrainment windows were
selected to allow sufficient time for plant growth and for cotyledons to
be imaged, but before the development of the first true leaves, which
can obscure cotyledon imaging.
We estimated period values through BioDare2 (Zielinski et al. 2014).
Values for period were determined by two algorithmic methods: MFourFit
(a curve-fitting method) and MESA (Maximum Entropy Spectral Analysis; an
autoregressive model based on stochastic modelling). Settings for both
analyses included removing estimates of period lower than 18 h and
higher than 30 h. Traces from experimental replicates that displayed no
rhythmicity of pattern were discarded. For each individual replicate
measurement, we compared the difference between the two analyses and
values that differed by more than 10 % (~2 h) were
removed. Statistical outliers for each family line within the
populations were removed. Mean values for each family line and
subsequently each population were then determined, as well as the
within-population range of values (the range from shortest to longest
circadian period family within each population).