Chlorophyll fluorescence of field-manipulated samples
In order to measure maximum PSII quantum efficiency
(F v/F m) and actual PSII
quantum efficiency (ΦPSII), chlorophyll fluorescence was carried out
according to a modified version of the protocol in (Clark 2020). Ten to
fifteen shoots of each specimen were sampled by selecting shoots
randomly from each cushion, when multiple cushions present. Shoots were
hydrated and quickly assembled into Hansatech FMS/LC dark-adaptation
leaf clips (Hansatech Instruments, Norfolk, UK) that were modified to
have a deeper cavity to allow tall moss shoots to stand upright on a
small, circular piece of filter paper created with a hole-punch. This
system allows the entire moss “bouquet” to be easily removed by
grabbing the filter paper with forceps, so that the same shoots could be
confidently measured at different time points.
Immediately after assembly of shoots in the clip, it was then closed to
allow the moss shoots to acclimate to darkness (allow PSII reaction
centers to open fully) for 30 minutes. At precisely 30 minutes, the clip
was placed into the Hansatech Pulse-Modulated chlorophyll fluorescence
measurement system (FMS) 2 (Hansatech Instruments, Norfolk, UK), the
clip was opened, and the script was run with actinic light of
photosynthetic flux density of 150 µmol photons m-2s-1 for 200 s and a saturation pulse of 3000 µmol
photons m-2 s-1 for 0.8 s before and
after the actinic light. Following the initial fluorometry time point (0
hours; T0), samples were placed into “water thrones”
as described in (Clark 2020). Water thrones are part of a system that
allows mosses to remain hydrated and near 100% RH through the water
wicking in a chemical laboratory wipe. The samples were placed in a
Percival E30B growth chamber (Percival Scientific, Perry, Iowa, USA)
under simulated winter recovery conditions consisting of a 10 hour
photoperiod (12 °C light, 5 °C dark) with 70 – 85% RH in chamber at
150 µmol m-2 s-1 where temperature
and RH were monitored with an iButton data logger (Maxim Integrated, San
Jose, CA, USA).
After 24 hours in recovery, samples were again placed into the
fluorometer clips, closed to allow PSII to dark acclimate, and run
through the fluorometer script for a second time point (24 hours;
T24). Following this round, samples were again
transferred to simulated winter recovery conditions in the growth
chamber water thrones until the next time point. Finally, fluorometer
measurements were repeated once more at 192 hours (eight days;
T192).
Fluorescence data were first tested for normality with the Shapiro-Wilk
test (Shapiro & Wilk 1965) and subsequently allF v/F m and ΦPSII were
compared pairwise using Wilcoxon signed-rank tests (Wilcoxon 1945) at
each time point. Significance was then adjusted for multiple-testing
with the Benjamini and Hochberg method (Benjamini & Hochberg 1995).