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).