About 450 million years ago, green algae belonging to the class Charophyceae emerged from their aquatic habitat to colonize the land [1–3]. This important event in the history of life gave rise to all the land plant species that make up the flora of our planet. The few thousand species of charophycean green algae that are alive today exhibit great variability in cellular organization and reproduction . With the land plants, they form the green plant lineage Streptophyta , whereas all other green algae (more than 10,000 species), with perhaps the exception of Mesostigma viride, belong to the sister lineage Chlorophyta . Five monophyletic groups of charophycean green algae have been recognized: the Chlorokybales, Klebsormidiales, Zygnematales, Coleochaetales and Charales , given here in order of increasing cellular complexity. Mesostigma may represent an additional lineage of the Charophyceae, the Mesostigmatales, as indicated by phylogenetic studies that placed this unicellular green alga at the base of the Streptophyta [7–10]. This lineage, however, remains controversial, considering that separate analyses based on a large number of chloroplast- or mitochondrial-encoded proteins [11–13] and on the chloroplast small and large subunit rRNA genes  identified Mesostigma before the divergence of the Chlorophyta and Streptophyta.
On the basis of morphological characters alone, the two charophycean groups that exhibit the greatest cellular complexity, i.e. the Charales and Coleochaetales, have been proposed to be the closest relatives of land plants [15, 16]. Recent analyses of the combined sequences of four genes from the nucleus (small subunit rRNA gene), chloroplast (atpB and rbcL) and mitochondria (nad5) of 25 charophycean green algae and eight green plants revealed that the Charales and land plants form a highly supported clade; however, moderate bootstrap support was observed for the positions of the other charophycean groups . The best trees inferred by Bayesian and maximum likelihood methods in this four-gene analysis support an evolutionary trend toward increasing cellular complexity . In contrast, all phylogenies of charophycean green algae previously inferred from a smaller number of genes failed to provide any conclusive results concerning the branching order of the charophycean green algae and their relationships with land plants [15, 16].
We have recently undertaken the sequencing of complete chloroplast genomes from representatives of the various charophycean lineages in order to elucidate the branching order of these lineages and also to understand the evolution of chloroplast DNA (cpDNA) within the Streptophyta. We have reported thus far the cpDNA sequences of Mesostigma (Mesostigmatales)  and Chaetosphaeridium globosum (Coleochaetales) . Comparative analyses of the Mesostigma cpDNA sequence (136 genes, no introns) with its land plant counterparts (110–120 genes, about 20 introns) revealed that the chloroplast genome underwent substantial changes in its architecture during the evolution of streptophytes (namely gene losses, intron insertions and scrambling of gene order). At the levels of gene content (125 genes), intron composition (18 introns) and gene order, Chaetosphaeridium cpDNA is remarkably similar to land plant cpDNAs, implying that most of the features characteristic of land plant lineages were acquired from charophycean green algae. Like the cpDNAs of many chlorophytes, those of Mesostigma, Chaetosphaeridium and most land plant species exhibit a quadripartite structure that is characterized by the presence of two copies of a rDNA-containing inverted repeat (IR) separated by large and small single-copy regions. All the genes they have in common, with a few exceptions, reside in corresponding genomic regions.
In this study, we report the complete cpDNA sequences of two members of the Zygnematales that belong to distinct lineages, Staurastrum punctulatum and Zygnema circumcarinatum. Although the chloroplast genomes of these charophycean green algae closely resemble their Chaetosphaeridium and bryophyte counterparts at the primary sequence and gene content levels, they feature substantial differences at the levels of structure, gene order and intron content. Like the cpDNA of the zygnematalean alga Spirogyra maxima , both Staurastrum and Zygnema cpDNAs lack a large IR. Clearly, loss of the IR appears to be a major event that shaped the architecture of the chloroplast genome in the Zygnematales, an event that apparently occurred early during the evolution of this group of charophycean green algae.