Land plants and green algae belonging to the Charophyceae sensu Mattox and Stewart form the Streptophyta, a lineage sister to the Chlorophyta which comprises most or all of the other green algae. A third green plant lineage, at the base of the split of the Chlorophyta and Streptophyta, is possibly represented by the green alga Mesostigma viride; however, this remains controversial because some phylogenetic analyses place Mesostigma within the Streptophyta. Five orders have been recognized in the Charophyceae: the Chlorokybales, Klebsormidiales, Zygnematales, Coleochaetales, and Charales. Based on its sarcinoid thallus, the only species in the Chlorokybales, Chlorokybus atmophyticus, has long been regarded as the earliest-diverging member of the Charophyceae. This rare species has been collected only in terrestrial habitats. In the present study, we have determined the complete chloroplast DNA (cpDNA) sequence (149,681 bp) of Chlorokybus to gain insight into its phylogenetic position and also to better understand how the chloroplast genome evolved in the Streptophyta. We have found that Chlorokybus cpDNA closely resembles Mesostigma cpDNA in showing a high degree of ancestral features. It displays a quadripartite structure, encodes 138 genes (only two extra genes compared to Mesostigma cpDNA), and contains a single intron (in trnL(UAA)). The gene order in Chlorokybus cpDNA is similar to that of Mesostigma cpDNA; we estimate that 14 inversions would be required to convert the gene order of one alga into that of the other. Phylogenetic analyses of combined chloroplast protein sequences from Mesostigma, Chlorokybus, other streptophytes, and a number of chlorophytes will be presented. Overall, our results support the idea that the Chlorokybales represent an early-diverging lineage.

Key words: Charophyceae, Chlorokybus atmophyticus, chloroplast genome, green algae, phylogeny, Streptophyta