A positive relationship between cell size and chloroplast genome size within chloroplast-bearing protists has been hypothesized in the past and shown in some case studies, but other factors influencing chloroplast genome size during the evolution of chlorophyte algae have been less studied. We study chloroplast genome size and GC content as a function of habitats and cell size of chlorophyte algae. The chloroplast genome size of green algae in freshwater, marine and terrestrial habitats was differed significantly, with terrestrial algae having larger chloroplast genome sizes in general. The most important contributor to these enlarged genomes in terrestrial species was the length of intergenic regions. There was no clear difference in the GC content of chloroplast genomes from the three habitats categories. Functional morphological categories also showed differences in chloroplast genome size, with filamentous algae having substantially larger genomes than other forms of algae, and foliose algae had lower GC content than other groups. Chloroplast genome size showed no significant differences among the classes Ulvophyceae, Trebouxiophyceae, and Chlorophyceae, but the GC content of Chlorophyceae chloroplast genomes was significantly lower than that of Ulvophyceae and Trebouxiophyceae. There was a certain positive relationship between chloroplast genome size and cell size for the Chlorophyta as a whole and within each of three major classes. Our data also confirmed previous reports that ancestral quadripartite architecture had been lost many times independently in Chlorophyta. Finally, the comparison of the phenotype of chlorophytes algae harboring plastids uncovered that most of the investigated Chlorophyta algae housed a single plastid per cell.

This study determined the transcriptomes of eight Oedogoniales species, including six species from Oedogonium and two species from Oedocladium to conduct phylotranscriptomic and evolutionary analyses. 155,952 gene families and 192 single-copy orthogroups were detected. Phylotranscriptomic analyses based on single-copy orthogroups were conducted using supermatrix and coalescent-based approaches. The phylotranscriptomic analysis results revealed that Oedogonium is polyphyletic, and Oedocladium clustered with Oedogonium. Together with the transcriptomes of the OCC clade in the public database, the phylogenetic relationship of the three orders (Oedogoniales, Chaetophorales, Chaetopeltidales) is discussed. The non-synonymous (dN) to synonymous substitution (dS) ratios of single-copy orthogroups of the terrestrial Oedogoniales species using a branch model of phylogenetic analysis by maximum likelihood were estimated, which showed that 92 single-copy orthogroups were putative rapidly evolving genes. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses results revealed that some of the rapidly evolving genes were associated with photosynthesis, implying that terrestrial Oedogoniales species experienced rapid evolution to adapt to terrestrial habitats. The phylogenetic results combined with evolutionary analyses suggest that the terrestrialization process of Oedogoniales may have occured more than once.