Redefining Chlorobotryaceae as one of the principal and most diverse lineages of eustigmatophyte algae.

Eustigmatophyceae is one of the ∼17 classes of the vast algal phylum Ochrophyta. Over the last decade, the eustigmatophytes emerged as an expansive group that has grown from the initially recognized handful of species to well over 200 genetically distinct entities (potential species). Yet the majority of eustigs, remain represented by unidentified strains, or even only metabarcode sequences obtained from environmental samples. Moreover, the formal classification of the group has not yet been harmonized with the recently uncovered diversity and phylogenetic relationships within the class. Here we make a major step towards resolving this issue by addressing the diversity, phylogeny and classification of one of the most prominent eustigmatophyte clades previously informally called the "Eustigmataceae group". We obtained 18S rDNA and rbcL gene sequences from four new strains from the "Eustigmataceae group", and from several additional eustig strains, and performed the most comprehensive phylogenetic analyses of Eustigmatophyceae to date. Our results of these analyses confirm the monophyly of the "Eustigmataceae group" and define its major subclades. We also sequenced plastid genomes of five "Eustigmataceae group" strains to not only improve our understanding of the plastid gene content evolution in eustigs, but also to obtain a robustly resolved eustigmatophyte phylogeny. With this new genomic data, we have solidified the view of the "Eustigmataceae group" as a well-defined family level clade. Crucially, we also have firmly established the genus Chlorobotrys as a member of the "Eustigmataceae group". This new molecular evidence, together with a critical analysis of the literature going back to the 19th century, provided the basis to radically redefine the historical concept of the family Chlorobotryaceae as the formal taxonomic rubric corresponding to the "Eustigmataceae group". With this change, the family names Eustigmataceae and Characiopsidaceae are reduced to synonymy with the Chlorobotryaceae, with the latter having taxonomic priority. We additionally studied in detail the morphology and ultrastructure of two Chlorobotryaceae members, which we describe as Neustupella aerophytica gen. et sp. nov. and Lietzensia polymorpha gen. et sp. nov. Finally, our analyses of partial genomic data from several Chlorobotryaceae representatives identified genes for hallmark flagellar proteins in all of these strains. The presence of the flagellar proteins strongly suggests that zoosporogenesis is a common trait of the family and also occurs in the members never observed to produce flagellated stages. Altogether, our work paints a rich picture of one of the most diverse principal lineages of eustigmatophyte algae.