Structure and formation of the perforated theca defining the Pelagophyceae (Heterokonta), and three new genera that substantiate the diverse nature of the class.

The pelagophytes, a morphologically diverse class of marine heterokont algae, have been historically united only by DNA sequences. Recently we described a novel perforated theca (PT) encasing cells from the Pelagophyceae and hypothesized it may be the first morphological feature to define the class. Here we consolidate that observation, describing a PT for the first time in an additional seven pelagophyte genera, including three genera new to science. We established clonal cultures of pelagophytes collected from intertidal pools located around Australia, and established phylogenetic trees based on nuclear 18S rDNA and plastid rbcL, psaA, psaB, psbA and psbC gene sequences that led to the discovery of three new species: Wyeophycus julieharrissiae and Chromopallida australis form a distinct lineage along with Ankylochrysis lutea within the Pelagomonadales, while Pituiglomerulus capricornicus is sister genus to Chrysocystis fragilis in the Chrysocystaceae (Sarcinochrysidales). Using fixation by high-pressure freezing for electron microscope observations, a distinctive PT was observed in the three new genera described in this paper, as well as four genera not previously investigated: Chrysoreinhardia, Sargassococcus, Sungminbooa and Andersenia. The mechanism of PT formation is novel, being fabricated from rafts in Golgi-derived vesicles before being inserted into an established PT. Extracellular wall and/or mucilage layers assemble exterior to the PT in most pelagophytes, the materials likewise secreted by Golgi-derived vesicles, though the mechanism of secretion is novel. Secretory vesicles never fuse with the plasma membrane as in classic secretion and deposition, but rather relocate extracellularly beneath the PT and disintegrate, the contents having to pass through the PT prior to wall and/or mucilage synthesis. This study substantiates the diverse nature of pelagophytes, and provides further evidence that the PT is a sound morphological feature to define the Pelagophyceae, with all 14 of the 20 known genera studied to date by TEM possessing a PT.