*2.3. Isolation of Symbiodiniaceae Cells*

From each of the 66 samples preserved in 100% ethanol, 1 cm fragments were cut, amounting to an estimated 20 mg of tissue for both species. Each fragment included about two polyps in the case of *S. maldivensis or* about eight polyps in the case of *C. abies.* Following a method adapted from [45] to isolate Symbiodiniaceae cells from cnidarians, samples were placed in 1.5 mL tubes with 500 μL of a 2 M sodium hydroxide solution. These were incubated at 37 ◦C for 1 h and then vortexed at a medium speed (using an Analitik Jena Tmix homogenizer) at the same temperature for about 2 h until complete lysis of the antipatharian tissues had occurred. The tubes were then centrifuged at 8000 rpm for 3 min, and the supernatant was discarded, then the pellet was resuspended in ultra-pure (Milli-Q) water and vortexed. One drop of lugol was added to the solution to re-stain the cells that might have lost pigments after preservation. About 8 μL of the pellet in 7 μL was used per chamber. Counts of visible Symbiodiniaceae were undertaken using a glass haemocytometer and an Axioscope A1 (Zeiss) light microscope. The number of the microalgae cells in all nine squares (1 mm2 each) of four counting chambers per sample was counted in order to estimate the density of cells per μL. To corroborate the efficiency of the procedure, the isolation protocol was also carried out on fresh and preserved (100% ethanol) samples of the scleractinian coral *Seriatopora hystrix* (Figure 3). To test for differences in microalgae density between antipatharian species, depths and colony region (top, middle or bottom part), a quasi-Poisson generalised linear model (GLM) was fitted using the 'stats' package in R [46]. The power to detect differences between depths was calculated using the function 'power.t.test' in the same 'stats' package.

**Figure 3.** Light microscopy images of Symbiodiniaceae-like extracts obtained through the sodium hydroxide isolation method. (**a**) Extract from the antipatharian *Cupressopathes abies* showing only one Symbiodiniaceae-like cell (arrow) but abundant cnidocytes. (**b**) Extract from the scleractinian *Seriatopora hystrix* showing numerous Symbiodiniaceae-like cells, corroborating the efficiency of the isolation method. Scale bars = 10 μm.


Histological sections were taken to determine the presence and location of Symbiodiniaceae cells within the coral's tissue. About 1.5 cm of each sample was dehydrated using increasing concentrations of ethanol, then soaked in butanol at 60 ◦C for 24 h. These samples were then embedded in liquid paraffin. Serial sections of 7 μm were cut using a Microm HM 340E (Zeiss) microtome and stained with a Masson's trichrome. The sections were observed and photographed using an Axioscope A1 (Zeiss) light microscope and Axiocam 305 camera.
