The complete chloroplast DNA sequences of the charophycean green algae Staurastrum and Zygnema reveal that the chloroplast genome underwent extensive changes during the evolution of the Zygnematales

Abstract

Background: The Streptophyta comprise all land plants and six monophyletic groups of charophycean green algae. Phylogenetic analyses of four genes from three cellular compartments support the following branching order for these algal lineages: Mesostigmatales, Chlorokybales, Klebsormidiales, Zygnematales, Coleochaetales and Charales, with the last lineage being sister to land plants. Comparative analyses of the Mesostigma viride (Mesostigmatales) and land plant chloroplast genome sequences revealed that this genome experienced many gene losses, intron insertions and gene rearrangements during the evolution of charophyceans. On the other hand, the chloroplast genome of Chaetosphaeridium globosum (Coleochaetales) is highly similar to its land plant counterparts in terms of gene content, intron composition and gene order, indicating that most of the features characteristic of land plant chloroplast DNA (cpDNA) were acquired from charophycean green algae. To gain further insight into when the highly conservative pattern displayed by land plant cpDNAs originated in the Streptophyta, we have determined the cpDNA sequences of the distantly related zygnematalean algae Staurastrum punctulatum and Zygnema circumcarinatum.

Results: The 157,089 bp Staurastrum and 165,372 bp Zygnema cpDNAs encode 121 and 125 genes, respectively. Although both cpDNAs lack an rRNA-encoding inverted repeat (IR), they are substantially larger than Chaetosphaeridium and land plant cpDNAs. This increased size is explained by the expansion of intergenic spacers and introns. The Staurastrum and Zygnema genomes differ extensively from one another and from their streptophyte counterparts at the level of gene order, with the Staurastrum genome more closely resembling its land plant counterparts than does Zygnema cpDNA. Many intergenic regions in Zygnema cpDNA harbor tandem repeats. The introns in both Staurastrum (8 introns) and Zygnema (13 introns) cpDNAs represent subsets of those found in land plant cpDNAs. They represent 16 distinct insertion sites, only five of which are shared by the two zygnematalean genomes. Three of these insertions sites have not been identified in Chaetosphaeridium cpDNA.

Conclusion: The chloroplast genome experienced substantial changes in overall structure, gene order, and intron content during the evolution of the Zygnematales. Most of the features considered earlier as typical of land plant cpDNAs probably originated before the emergence of the Zygnematales and Coleochaetales.

Figure 1

Gene maps of Staurastrum and Zygnema cpDNAs. Genes (filled boxes) shown on the outside of each map are transcribed in a clockwise direction, whereas those on the inside of each map are transcribed counterclockwise. Genes absent from Marchantia cpDNA are represented in beige. Gene clusters shared with Marchantia cpDNA [GenBank:NC_001319] are shown as alternating series of green and red boxes. Genes present in Marchantia cpDNA but located outside conserved clusters are shown in grey. Gene clusters shared by the two zygnematalean cpDNAs are represented by labelled bars outside each map. Genes containing introns (open boxes) are denoted by asterisks. Dispersed repeat regions in Zygnema cpDNA that contain short tandem repeats are denoted by symbols. The repeat units in these regions are as follows: filled squares, TAGAA; open squares, TTCTA; filled circles, GTAT; open circles, ATAC; filled triangles, CTTA. Note that filled and open symbols with the same geometric shape represent the repeat regions of which the sequences are in inverted orientation relative to one another. The intron sequences bordering the rps12 exons (rps12a and rps12b) are spliced in trans at the RNA level. tRNA genes are indicated by the one-letter amino acid code (Me, elongator methionine; Mf, initiator methionine) followed by the anticodon in parentheses. The ORFs unique to Staurastrum or Zygnema cpDNA are not indicated (see [GenBank:AY958085] and [GenBank:AY958086] for more details).

Figure 2

Fragmentation of ancestral chloroplast gene clusters during the evolution of the Zygnematales. The ancestral clusters shown are found in both Mesostigma [GenBank:NC_002186] and Marchantia [GenBank:NC_001319] cpDNAs. The top and bottom arrows denote the sites where they are broken in Staurastrum and Zygnema cpDNAs, respectively. For the polarities of the genes relative to one another, the reader should consult the gene map of Mesostigma cpDNA [11].

Figure 3

Compared patterns of gene partitioning in zygnematalean and Marchantia cpDNAs. Each gene in Staurastrum and Zygnema cpDNAs is colour-coded according to the region of Marchantia cpDNA [GenBank:NC_001319] carrying its homologue; cyan, large single-copy region; magenta, small single-copy region; and yellow, IR. Genes shown in grey are absent from Marchantia cpDNA.

Figure 4

Distributions of introns in streptophyte cpDNAs. Circles denote the presence of group I introns, and squares denote the presence of group II introns. Divided squares represent trans-spliced group II introns. Open symbols denote the absence of intron ORFs, whereas filled symbols denote their presence. Intron insertion sites in protein-coding and tRNA genes are given relative to the corresponding genes in Mesostigma cpDNA; the insertion site in rrl is given relative to the Escherichia coli 23S rRNA. For each insertion site, the position corresponding to the nucleotide immediately preceding the intron is reported. Note that rps16 is lacking in Marchantia cpDNA and that the rrl intron at position 2593 is absent from all completely sequenced land plant cpDNAs, with the exception of Anthoceros cpDNA. The intron data were taken from the following accession numbers: Staurastrum, [GenBank:AY958085]; Zygnema, [GenBank:AY958086]; Chaetosphaeridium, [GenBank:NC_004115]; Marchantia, [GenBank:NC_001319]; and Anthoceros formosae [GenBank:NC_004543].

Figure 5

Sequence conservation among streptophyte MatK proteins. The MatK sequences of selected green algae and land plants were aligned with T-COFFEE [40] and arranged into two separate groups. Identical amino acids in all the sequences examined are displayed on a black background, whereas identical amino acids in all the green algal or land plant sequences are shown on a dark grey background. In each group, sets of residues sharing eight of the 10 features in the property matrix of AMAS [41] are shown on a light grey background. The accession numbers for the MatK sequences analyzed are as follows: Zygnema, [GenBank:AY958086]; Staurastrum, [GenBank:AY958085]; Chaetosphaeridium, [GenBank:NC_004115]; Chara connivens, [GenBank:AY170442]; Nitella opaca, [GenBank:AY170449]; Marchantia, [GenBank:NC_001319]; and Physcomitrella patens [GenBank:NC_005087].