A clade uniting the green algae Mesostigma viride and Chlorokybus atmophyticus represents the deepest branch of the Streptophyta in chloroplast genome-based phylogenies

Background: The Viridiplantae comprise two major phyla: the Streptophyta, containing the charophycean green algae and all land plants, and the Chlorophyta, containing the remaining green algae. Despite recent progress in unravelling phylogenetic relationships among major green plant lineages, problematic nodes still remain in the green tree of life. One of the major issues concerns the scaly biflagellate Mesostigma viride, which is either regarded as representing the earliest divergence of the Streptophyta or a separate lineage that diverged before the Chlorophyta and Streptophyta. Phylogenies based on chloroplast and mitochondrial genomes support the latter view. Because some green plant lineages are not represented in these phylogenies, sparse taxon sampling has been suspected to yield misleading topologies. Here, we describe the complete chloroplast DNA (cpDNA) sequence of the early-diverging charophycean alga Chlorokybus atmophyticus and present chloroplast genome-based phylogenies with an expanded taxon sampling. Results: The 152,254 bp Chlorokybus cpDNA closely resembles its Mesostigma homologue at the gene content and gene order levels. Using various methods of phylogenetic inference, we analyzed amino acid and nucleotide data sets that were derived from 45 protein-coding genes common to the cpDNAs of 37 green algal/land plant taxa and eight non-green algae. Unexpectedly, all best trees recovered a robust clade uniting Chlorokybus and Mesostigma. In protein trees, this clade was sister to all streptophytes and chlorophytes and this placement received moderate support. In contrast, gene trees provided unequivocal support to the notion that the Mesostigma + Chlorokybus clade represents the earliest-diverging branch of the Streptophyta. Independent analyses of structural data (gene content and/or gene order) and of subsets of amino acid data progressively enriched in slow-evolving sites led us to conclude that the latter topology reflects the true organismal relationships. Conclusion: In disclosing a sister relationship between the Mesostigmatales and Chlorokybales, our study resolves the long-standing debate about the nature of the unicellular flagellated ancestors of land plants and alters significantly our concepts regarding the evolution of streptophyte algae. Moreover, in predicting a richer chloroplast gene repertoire than previously inferred for the common ancestor of all streptophytes, our study has contributed to a better understanding of chloroplast genome evolution in the Viridiplantae.