Seven newly sequenced chloroplast genomes from the order Watanabeales (Trebouxiophyceae, Chlorophyta): Phylogenetic and comparative analysis.

The little-known order Watanabeales currently includes 10 genera with Chlorella-like species that reproduce by unequal-sized autospores and are predominantly solitary or terrestrial. The taxonomic scheme of Watanabeales has only been primarily inferred by short and less informative rDNA phylogenetic analysis. In the present study, seven newly sequenced genomes and one reported chloroplast genome representing the existing major branches of Watanabeales were harvested to phylogenetically reconstruct this order and to further understand its evolution. All chloroplast genomes of Watanabeales ranging from 133 to 274 kb were circular mapping and lacked a quadripartite structure. The chloroplast genome size, GC content, number of introns, and length of intergenic region proportion of the Watanabeales showed consistent trends, with Calidiella yingdensis D201 and Kalinella pachyderma 2601 having the lowest and highest values, respectively, echoing the positive correlation between organismal size and genome size. Phylogenetic analysis of Watanabeales based on 76 protein-coding genes coupled with the establishment of various complex analytical methods determined the unique robust taxonomic scheme which was incongruent with rDNA. Comparative genomic analyses revealed that the chloroplast genomes of Watanabeales accounted for numerous complex rearrangements and inversions which indicated high cryptic diversity. Substitution rate estimation indicated that the chloroplast genomes of Watanabeales were under purifying selection and similar evolutionary pressure and supported the view that genus Symbiochloris should be excluded from Watanabeales. Our results enrich the chloroplast genome resources of Watanabeales, clarify the phylogenetic status of species within this order, and provide more reference information for subsequent taxonomic and phylogenetic study.