*4.1. Overall Analysis Results*

Despite photosymbiosis being evolutionarily conserved within most lineages in the subclass Hexacorallia [38], an endosymbiotic association between antipatharians and Symbiodiniaceae has not been clearly established. High densities of dinoflagellates (~10<sup>7</sup> cells cm–2) were found in three whip-like colonies (*Cirrhipathes* sp.) [37]. Therefore, it was considered that a study involving a higher number of samples was required in order to assess the microalgal presence and estimate their densities in a greater number of samples from more species and to identify possible patterns regarding species, colony morphology and depth. The present study showed either none or only very low densities (0–4 cells mm<sup>−</sup>3) of Symbiodiniaceae-like cells in both the whip-like *S. maldivensis* and the bushy *C. abies*, both on shallow and on mesophotic reefs. These new findings align with both previous historical observations and more subsequent studies that found few, if any, endosymbiotic algae in antipatharians [32–36]. These studies—ours included—evidence that high abundances of dinoflagellates within antipatharians is not a common finding. Considering the relevance of antipatharians for habitat provision on reefs and the increasing extent of mass bleaching events affecting scleractinian corals, the possibility of most antipatharians being exempt from this threat warrants further investigation.

While, in our study, the dinoflagellates' identity could not be determined by molecular analysis, the Symbiodiniaceae-like cells observed in the histological sections of the whip-like *S. maldivensis* closely resembled those described in the other two studies [34,37] (Figure 4). For future histological examination of antipatharians, where the tissue cannot be separated from the skeleton (such as *C. abies*), we suggest experimenting with softening the skeletons with a lytic polysaccharide monooxygenases (LPMOs) treatment [49]. We applied the same sodium hydroxide isolation protocol—a protocol found to be effective for scleractinians and other cnidarians [45]—to a scleractinian coral fragment, and it was clear that the cells obtained were Symbiodiniaceae (Figure 3). Nevertheless, it is probably safer to refer to the cells in our antipatharian samples only as being Symbiodiniaceae-like.

In the present study, Symbiodiniaceae-like cells were observed in the SEM and histological examination located inside the coral gastrodermis of *S. maldivensis* (Figures 5 and 6). This suggests an endosymbiotic association, which was also suggested from histological sections analysis and from ultrastructural analysis [34,37]. The fact that no Symbiodiniaceae ITS2 sequences were amplified from the DNA extractions in this study seems most likely due to the extremely low density of dinoflagellates. In the two studies where identification was possible, different genera (*Cladocopium*, *Gerakladium* and *Durusdinium*) were reported to be associated with different antipatharian species [34,37]. Such plasticity, even at the intra-colony level, has also been evidenced in more recent studies on scleractinian corals and is believed to be environmentally driven [24]. Moreover, there is evidence to suggest that the same Symbiodiniaceae species may be mutualistic in one host context but opportunistic in another [50]. Therefore, the identity of the dinoflagellates alone is insufficient to determine the type of symbiosis. Further studies will be necessary to determine the nature of the association between Symbiodiniaceae and antipatharians, particularly in cases where microalgae abundance is high, as in [37].
