Heat-evolved coral photosymbionts exhibit dampened stress responses across distinct physiological contexts.

Experimental evolution under elevated temperatures has generated heat-evolved (HE) strains of Symbiodiniaceae that enhance coral bleaching tolerance. However, the biomolecular mechanisms underlying this resilience remain poorly understood. We conducted a laboratory heat-stress experiment and applied synchrotron-based Fourier transform infrared (FTIR) microspectroscopy to examine physiological and biomolecular responses of HE (strain: SS8) and wild-type (strain: WT10) Cladocopium proliferum to thermal stress across three physiological contexts: in hospite, expelled, and cultured. In hospite, both strains exhibited heat-induced increases in free amino acids, phosphorylated compounds, and lipids, coupled with reduced protein content - hallmarks of cellular stress. SS8, however, showed a dampened response overall, in line with an improved thermotolerance based on holobiont phenotypes. Expelled and in hospite cells shared broadly similar biomolecular profiles, though expelled cells of both strains responded less strongly - indicating expulsion may relieve host-imposed stress. Cultured cells differed from in hospite and expelled cells but showed similar strain-specific trends. WT10 responded strongly to heat stress - displaying depleted amino acids, phosphorylated metabolites, and disrupted lipid balance - whereas SS8 mounted a relatively muted metabolic response. These findings support the potential of HE symbionts for reef restoration, highlight the importance of physiological context in assessing Symbiodiniaceae thermotolerance, and the utility of single-cell FTIR microspectroscopy.