Validation of parameters and protocols derived from chlorophyll a fluorescence commonly utilised in marine ecophysiological studies.

This study documents the first validation of the suitability of the most common parameters and protocols used in marine ecophysiology to characterise photosynthesis by means of chlorophyll a fluorescence tools. We demonstrate that the effective yield of PSII (ΔF /F m ') is significantly underestimated when using short inductions times (≤1 min) following the rapid light curve protocol (RLC). The consequent electron transport rates (ETR) underestimations are species-specific and highly variable with irradiance and the photoacclimatory condition of the sample. Our analysis also questions the use of relative descriptors (relETR), as they not only overestimate photosynthesis, but overlook one of the fundamental components of the photosynthetic response: light absorption regulation. Absorptance determinations were fundamental to characterise the ETR response of low-pigmented seagrass leaves, and also uncovered relevant differences between two coral species and the accclimatory response of a cultured dinoflagellate to temperature. ETR and oxygen evolution determinations showed close correlations for all organisms tested with the expected slope of 4 e- per O2 molecule evolved, when correct photosynthesis inductions and light absorption determinations were applied. However, ETR curves cannot be equated to conventional photosynthetic response to irradiance (P vs E ) curves, and caution is needed when using ETR to characterise photosynthesis rates above photosynthesis saturation (E k ). This validation strongly supports the utility of fluorescence tools, underlining the need to correct two decades of propagation of erroneous concepts, protocols and parameters in marine eco-physiology. We aim also to emphasise the importance of optical descriptions for understanding photosynthesis, and for interpreting fluorescence measurements. In combination with conventional gross photosynthesis (GPS) approaches, optical characterisations open an extraordinary opportunity to determine two central parameters of photosynthesis performance: the quantum yield (φmax ) of the process and its minimum quantum requirements (1/φmax ). The combination of both approaches potentiates the possibilities of chlorophyll a fluorescence tools to characterise marine photosynthesis biodiversity.