Appendix 1

Application of PAM Flurometry to cyanobacteria

An important consideration when assessing the photophysiology of cyanobacteria and other photosynthetic organisms is the contribution of phycobilins and PSI chlorophyll pigments to overall fluorescence. This complicates the interpretation of certain photosynthetic efficiency and non-photochemical quenching metrics (Fv/FM, ∆F/FM′, qN, NPQ) for cyanobacteria as they assume that the fluorescence signal is predominantly from PSII. Another key difference is the short term and long term state transitions that occur in cyanobacteria that regulate the distribution of excitation energy between PSII and PSI (Fujita et al. 1994). In plants, state transitions have only minor influences on the fluorescence of PSII (Krause and Weis 1991) and nonphotochemical quenching is dominated by thermal dissipation (Adams and Demmig-Adams 1993). However, in cyanobacteria non-photochemical quenching is mainly a reflection of short term state transitions from PSII – PSI to distribute excitation energy (Campbell et al. 1998). Long term state transitions also need to be considered when defining photoacclimation, as cyanobacteria grown in green-rich light environments will show a shift of excitation energy to PSI whereas cyanobacteria grown in red-rich light environments will show a shift of excitation energy to PSII (Fujita et al. 1994; Campbell 1996). In spite of these differences, multiple studies have shown that Electron Transport Rates (ETR) for PSII calculated by performing Rapid Light Curves (RLCs), using a PAM fluorometer, show a linear relationship with O2 evolution for within species comparisons over a range irradiances (Kromkamp et al. 2001; Masojídek et al. 2001) and in high light and low light conditions (Bañares‐España et al. 2013). These linear relationships support that the ETR of PSII from RLCs provides an appropriate relative measure of photosynthetic rate for comparisons of a single cyanobacterial species in different environments. Also by using the gradient of the initial linear increase of ETR with applied photosynthetically active radiation (PAR) during RLCs, in conjunction with the maximum ETR (ETRmax) the light saturation coefficient, Ek, can be calculated. The Ek of a photosynthetically active organism, which is photoacclimated, should be approximately the same as the maximum ambient PAR intensity experienced in its environment, as it represents its maximum rate of PSII photosynthesis (Ryan et al. 2004). In addition to these metrics a measurement of photochemical quenching, qP, has been shown to be an accurate representation of the proportion of open reaction centres for cyanobacteria, even though its trend with increasing irradiances for cyanobacteria appears to be different to that of plants (Campbell et al. 1998).

Appendix 2

Dark adaptation trials

Prior to commencing any of the RLCs, dark adaption trials were performed (see supplementary information). Campbell et al. (1998) found 5 minutes to be an appropriate dark adaption time for nine different strains of cyanobacteria, and this has been used in multiple cyanobacterial fluorometry studies since (e.g. Li et al. 2002; Ferroni et al. 2010). To test whether five minutes was suitable for O. spongeliae, the quantum yield of PS II was measured initially in the light and then after sequential and increasing periods of dark adaptation (1, 2, 3, 4, 5 and 10 mins) with three minutes of light between each recording. Dark adaptation was achieved by placing the fibre optic cable on the L. herbacea tissue, covering the L. herbacea and the cable with a black plastic bag and holding it in position for the length of time required, before taking the reading. For dark adaptation, the cable was moved to a different position on the L. herbacea for each successive period of dark adaptation and quantum yield measurement. Dark adaptation trials were performed on three L. herbacea from each reef site, which were selected in a randomly stratified manor with a minimum horizontal distance of 20 m apart across the reef site. During dark adaptation trials the mean effective quantum yield of L. herbacea from both reef sites decreased with increasing periods of dark adaptation up to a period of 5 minutes, where the mean effective quantum yield appeared to plateau. No significant difference was observed between the curves for these reef sites (GLMSite: F(1,4) = 0.267, p = 0.632) with the length of dark adaptation as a covariate and individual sponges nested within site as a random factor in the model.

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