Divergent copies of the large inverted repeat in the chloroplast genomes of ulvophycean green algae

Abstract

The chloroplast genomes of many algae and almost all land plants carry two identical copies of a large inverted repeat (IR) sequence that can pair for flip-flop recombination and undergo expansion/contraction. Although the IR has been lost multiple times during the evolution of the green algae, the underlying mechanisms are still largely unknown. A recent comparison of IR-lacking and IR-containing chloroplast genomes of chlorophytes from the Ulvophyceae (Ulotrichales) suggested that differential elimination of genes from the IR copies might lead to IR loss. To gain deeper insights into the evolutionary history of the chloroplast genome in the Ulvophyceae, we analyzed the genomes of Ignatius tetrasporus and Pseudocharacium americanum (Ignatiales, an order not previously sampled), Dangemannia microcystis (Oltmannsiellopsidales), Pseudoneochloris marina (Ulvales) and also Chamaetrichon capsulatum and Trichosarcina mucosa (Ulotrichales). Our comparison of these six chloroplast genomes with those previously reported for nine ulvophyceans revealed unsuspected variability. All newly examined genomes feature an IR, but remarkably, the copies of the IR present in the Ignatiales, Pseudoneochloris, and Chamaetrichon diverge in sequence, with the tRNA genes from the rRNA operon missing in one IR copy. The implications of this unprecedented finding for the mechanism of IR loss and flip-flop recombination are discussed.

Figure 1

Sequence coverages in the 15 ulvophycean chloroplast genomes compared in this study. (a) Sizes of the SSC, IR and LSC regions. Red arrows indicate the direction of transcription of the rRNA operon in IR-containing genomes. Genomes lacking the IR are represented in grey. The names of the newly examined taxa are indicated in red. (b) Amounts of coding, intronic, intergenic and small repeated sequences (≥30 bp). Note that intron-encoded genes were not considered as coding sequences but rather as intron sequences. The phylogenetic relationships among the taxa examined are derived from Fig. 2b.

Figure 2

Chloroplast phylogenomic trees of chlorophytes inferred from the PCG-AA and PCG12RNA data sets using RAxML and PhyloBayes. (a) Relationships among the major lineages of the Chlorophyta. Strongly supported clades are represented as triangles with sizes proportional to the number of taxa (indicated in parentheses). Bootstrap support and posterior probability values are reported on the nodes. (b) Relationships among ulvophycean taxa. The best-scoring RAxML tree inferred from the PCG12RNA data set is presented. Bootstrap support values are reported on the nodes: from top to bottom are shown the values for the RAxML analyses of the PCG12RNA and PCG-AA data sets. The names of the newly examined taxa are indicated in red.

Figure 3

Gene partitioning patterns of ulvophycean chloroplast genomes. The suite of genes in each IR-containing genome is displayed so that the SC region with the gene content the most similar to that predicted for the ancestral SSC region of core chlorophytes is presented at the bottom of the figure. Thick vertical lines delimit the genes encoded in the IR (thick black lines, identical IR copies; thick brown lines, divergent IR copies). The genes making up the rDNA operon are highlighted in yellow whereas those present in the SSC region of Trichosarcina are highlighted in blue. Red letterings designate the genes of ancestral LSC origin that have been acquired by the IRs of core chlorophytes.

Figure 4

Rearrangements of gene order among ulvophycean chloroplast genomes. The numbers of gene reversals were estimated using MGR v2.03 and the tree topology shown in Fig. 2b. The gene order data set contained 98 genes and included three conserved genes carrying frameshift mutations in Bryopsis hypnoides (rpoB, rpoC1 and ycf20). The names of the newly examined taxa are indicated in red.

Figure 5

Comparison of IR sequences in the ulvophycean chloroplast genomes carrying non-identical IR copies. Regions displaying similar sequences are connected by shaded areas, with sequence identity denoted by the grey scale. Red and dark blue boxes represent coding regions of rRNA and tRNA genes, respectively; turquoise and white boxes represent ORFs and noncoding regions within introns, respectively.

Figure 6

Analysis of flip-flop recombination in the Ignatius (a) and Pseudoneochloris (b) chloroplast genomes. PCR assays using eight different pairs of primers were carried out to test whether the non-identical IRs in each algal genome undergo homologous recombination. Primer locations and polarities are indicated by numbered arrows on the diagrams showing the organizations of the IR copies (see Supplementary Table S3 for the primer sequences). PCR products were analyzed by electrophoresis on agarose gels; the numbers above the gel lanes indicate the combinations of primers used.

Figure 7

Introns in ulvophycean chloroplast genomes. (a) Distribution of group I introns. (b) Distribution of group II introns. A grey box denotes an intron lacking an ORF, whereas a colored box represents an intron containing an ORF (see the color code for the type of intron-encoded protein). Stars denote the intron insertion sites that have not been observed in other groups of chlorophytes. Intron insertion sites in protein-coding and tRNA genes are given relative to the corresponding genes in the Mesostigma chloroplast genome; insertion sites in rrs and rrl are given relative to E. coli 16S and 23S rRNAs, respectively. For each insertion site, the position corresponding to the nucleotide immediately preceding the intron is reported. Abbreviations: EN, H-N-H endonuclease; RT, reverse transcriptase; X, intron maturase. (c) Phylogenetic relationships among group II introns of the Ignatiales, Oltmannsiellopsidales, Ulvales and Ulotrichales. The tree shown here was inferred by RAxML analysis of an alignment of 124 nucleotides corresponding to domains IA, IVB, V and VI of the core secondary structure. Bootstrap support values higher than 50% are reported on the nodes. Colored circles denote the types of intron ORF (see the color code on panel b). Note that clades I through IV were identified in a previous phylogenetic study of Gloeotilopsis group II introns. Names of the taxa newly examined in the present investigation are indicated in red. Abbreviations: Cc, Chamaetrichon capsulatum; Dm, Dangemannia microcystis; Gp, Gloeotilopsis planctonica; Gs, Gloeotilopsis sarcinoidea; It, Ignatius tetrasporus; Pm, Pseudoneochloris marina; Tm, Trichosarcina mucosa; Us, Ulva sp. UNA00071828.