Quantifying the uncertainties in multi-wavelength PAM fluorometry due to innate and irradiance-induced variability of fluorescence spectra

Multi-wavelength Chl a fluorometers are increasingly applied to assess phytoplankton photosynthetic capacity and composition, but their usefulness is limited by uncertainties in fluorescence excitation spectra (FES). We investigated this issue using the Phyto-PAM fluorometer to evaluate the effects of innate and irradiance-dependent variations in background (F) and variable (Fv) FES on analysis of three pigment groups (cyanobacteria, chlorophytes and chromophytes). The effects on group-specific estimates of minimum fluorescence (F0), a proxy for biomass, and Fv/Fm, the quantum yield of photochemistry, presented some challenges to the interpretation of group-specific results. F0 estimates usually had a 5–15% margin of error, even when measuring highly uneven mixtures, and applying imperfectly matched calibration FES or stressing samples with photosynthetically active and ultraviolet radiation; errors in Fv/Fm were commonly < 15%. Despite such relatively good accuracy, estimates for F0 and, especially, Fv/Fm are unreliable for groups at low relative abundance, and results can sometimes be reported for groups not actually present. We report margins of error for different levels of relative abundance to inform interpretation of measurements from natural communities and show that F and Fv spectra for some taxa can differ in ways that produce severe errors in F0 and Fv/Fm estimates if used uncritically.