*2.2. The Cell Morphology and Ultrastructure*

Several morphological types of cells were found in BM1 cultures. The first cell type was represented by motile spherical zoospores (15–20 ΐm in diameter with a mucous sheath) with two isokont anterior flagellae and a discoid eyespot near the cytoplasmic membrane at the cell anterior (Figure 2a). The zoospores featured a cupshaped chloroplast occupying almost the entire cell volume. Another type was comprised by non-motile coccoid cells (20–40 ΐm in diameter; Figure 2b). The cells contained a centrally located spherical nucleus. Palmelloid clusters of two to eight cells were formed occasionally. Under adverse environmental conditions, the coccoid cells increased in size (up to 80 ΐm) and accumulated red-colored spherical inclusions in the cytoplasm, which tended to cluster around the nucleus (Figure 2c). The red inclusions gradually occupied the entire cytoplasm volume resulting in the formation of resting "red" cells (Supplementary Figure S2a). 

**Figure 2.** Life cycle stages of BM1 isolate (**a**) Zoospore; ( **b**) Coccoid green cell; (**c**) Coccoid green cell with red-colored lipid droplets; ( **d**) Sporangium; (**e**) Putative isogamous sexual process. 

The isolated microalgae propagated mainly via asexual reproduction forming sporangia containing two to eight autospores (Figure 2d). After shedding the sporangium wall, the newly formed cells remained attached to each other for a long time; as a result, the culture tended to accumulate four-cell clusters. Under nonoptimal cultivation conditions, e.g., in a highly diluted culture, small, pear-shaped fast-moving biflagellate cells similar to gametes as described by Triki *et al.* [15] were encountered. Occasionally, these cells underwent conjugation resembling isogamous sexual process (Figure 2e). Dead cells with transparent content were also present in the culture (not shown). One could conclude that the life cycle and cell morphology recorded in the BM1 isolate as well as the ability to accumulate the red pigment in the "red" cells are consistent with the characteristic traits of *Haematococcus pluvialis* Flotow [15–17]. 

To characterize the newly isolated microalga, we investigated its cell ultrastructure. It should be noted that ultrastructural studies of *H. pluvialis* are generally more difficult in comparison to most of green microalgae, mainly due to the presence of tough cell walls complicating the chemical fixation, embedding and preparation of ultrathin sections [18]. Indeed, we found that the thick cell wall of BM1 was, like that of *H. pluvialis* aplanospores, extremely resistant to mechanical disruption and chemical agents and presented difficulties for electron microscopy. Nevertheless, both transmission (Figure 3a,c,d) and scanning (Figure 3b) electron micrographs of "green" and "red" BM1 cells were obtained. 

**Figure 3.** Electron micrographs of *H. pluvialis* BM1: (**a**) transmission electron microscopy (TEM) of a "green" cell; ( **b**) scanning electron microscopy (SEM) of enrichment culture comprised of different cell types; (**c**) TEM of a "red" cell; ( **d**) Pyrenoid structure typical of BM1 cells. CW— cell wall; LD—lipid droplets; SG—starch grains; T—thylakoids. Note the absence of LD in the "green" cells (**a**) and their presence in the "red" cells (**c**). 

As was shown by electron microscopy, the BM1 cells at different stages of life cycle were spherical and 18–59 ΐm in diameter (Figure 3b). Green flagellated and palmelloid cells possessed a thick (0.64–0.8 ΐm) extracellular matrix, which was thinner in the palmelloid and "red" cells. All kinds of palmelloid cells formed (up to 0.4 ΐm) the thick cell wall. The chloroplast contained two to ten pyrenoids and a few starch grains (Figure 3a,d). In the resting "red" cells, a pronounced decrease in the thylakoid volume and number was recorded; large merging lipid droplets were also present, which (Figure 3c) eventually occupied almost the entire cell volume. 
