Quantifying pigment content in crustose coralline algae using hyperspectral imaging: A case study with Tethysphytum antarcticum (Ross Sea, Antarctica)

Crustose coralline algae (CCA) are a highly diverse group of habitat-forming, calcifying red macroalgae (Rhodophyta) with unique adaptations to diverse irradiance regimes. A distinctive CCA phenotype adaptation, which allows them to maximize photosynthetic performance in low light, is their content of a specific group of light-harvesting pigments called phycobilins. In this study, we assessed the potential of noninvasive hyperspectral imaging (HSI) in the visible spectrum (400-800 nm) to describe the phenotypic variability in phycobilin content of an Antarctic coralline, Tethysphytum antarcticum (Hapalidiales), from two distinct locations. We validated our measurements with pigment extractions and spectrophotometry analysis, in addition to DNA barcoding using the psbA marker. Targeted spectral indices were developed and correlated with phycobilin content using linear mixed models (R² = 0.64-0.7). Once applied to the HSI, the models revealed the distinct phycoerythrin spatial distribution in the two site-specific CCA phenotypes, with thin and thick crusts, respectively. This study advances the capabilities of hyperspectral imaging as a tool to quantitatively study CCA pigmentation in relation to their phenotypic plasticity, which can be applied in laboratory studies and potentially in situ surveys using underwater hyperspectral imaging systems.