Seven chloroplast genomes and morphological data clarify the phylogenetic position and plastome evolution of Characium-like green algae

Characium and Characium -like algae represent a taxonomically challenging group within Chlorophyceae. Due to their high phenotypic plasticity and convergent morphology, their true evolutionary relationships have long remained unresolved, and the lack of genomic resources has further hindered taxonomic revision. We sequenced and assembled seven complete chloroplast genomes from Characium and morphologically similar taxa and documented their detailed cell morphology. Comparative genomic analyses revealed that while most genomes retained the typical quadripartite structure, they exhibited significant structural variation, including gene rearrangements and diverse IR expansions/contractions. Furthermore, phylogenetic analyses based on 64 protein-coding genes successfully resolved the long-standing taxonomic ambiguity of Characium and Characium -like algae, placing the strains into three distinct, well-supported clades within Sphaeropleales and Volvocales. The synteny patterns and DCJ distances were consistent with the phylogeny: closely related species showed largely conserved gene order and low DCJ distances, whereas more distant lineages exhibited extensive rearrangements and higher distances. Branch-site tests identified a small set of positively selected genes, mainly related to photosynthesis, proteolysis, and plastid gene expression, suggesting that adaptive processes may have contributed to chloroplast genome diversity in these algae. This study establishes the first clear phylogenomic framework for these lineages, revealing distinct evolutionary patterns of gene loss and structural rearrangement that align with the newly resolved clades. It also provides an important basis for advancing studies on chloroplast genome evolution within Chlorophyceae.