Mitochondrial genomes (mitogenomes) of flowering plants are well known for their extreme diversity in size, structure, gene content, and rates of sequence evolution and recombination. In contrast, little is known about mitogenomic diversity and evolution within gymnosperms. Only a single complete genome sequence is available, from the cycad Cycas taitungensis, while limited information is available for the one draft sequence, from Norway spruce (Picea abies). To examine mitogenomic evolution in gymnosperms, we generated complete genome sequences for the ginkgo tree (Ginkgo biloba) and a gnetophyte (Welwitschia mirabilis). There is great disparity in size, sequence conservation, levels of shared DNA, and functional content among gymnosperm mitogenomes. The Cycas and Ginkgo mitogenomes are relatively small, have low substitution rates, and possess numerous genes, introns, and edit sites; we infer that these properties were present in the ancestral seed plant. By contrast, the Welwitschia mitogenome has an expanded size coupled with accelerated substitution rates and extensive loss of these functional features. The Picea genome has expanded further, to more than 4 Mb. With regard to structural evolution, the Cycas and Ginkgo mitogenomes share a remarkable amount of intergenic DNA, which may be related to the limited recombinational activity detected at repeats in Ginkgo Conversely, the Welwitschia mitogenome shares almost no intergenic DNA with any other seed plant. By conducting the first measurements of rates of DNA turnover in seed plant mitogenomes, we discovered that turnover rates vary by orders of magnitude among species.

Using an independent fosmid cloning approach and comprehensive transcriptome analysis to complement data from the Selaginella moellendorffii genome project, we determined the complete mitochondrial genome structure of this spikemoss. Numerous recombination events mediated mainly via long sequence repeats extending up to 7kbp result in a complex mtDNA network structure. Peculiar features associated with the repeat sequences are more than 80 different microsatellite sites (predominantly trinucleotide motifs). The S. moellendorffii mtDNA encodes a plant-typical core set of a twin-arginine translocase (tatC), 17 respiratory chain subunits, and 2 rRNAs but lacks atp4 and any tRNA genes. As a further novelty among plant chondromes, the nad4L gene is encoded within an intron of the nad1 gene. A total of 37 introns occupying the 20 mitochondrial genes (four of which are disrupted into trans-splicing arrangements including two novel instances of trans-splicing introns) make the S. moellendorffii chondrome the intron-richest and gene-poorest plant mtDNA known. Our parallel transcriptome analyses demonstrates functional splicing of all 37 introns and reveals a new record amount of plant organelle RNA editing with a total of 2,139 sites in mRNAs and 13 sites in the two rRNAs, all of which are exclusively of the C-to-U type.

Plant mitochondrial genomes show much more evolutionary plasticity than those of animals. We analysed the first mitochondrial DNA (mtDNA) of a lycophyte, the quillwort Isoetes engelmannii, which is separated from seed plants by more than 350 million years of evolution. The Isoetes mtDNA is particularly rich in recombination events, and chloroplast as well as nuclear DNA inserts document the incorporation of foreign sequences already in this most ancestral vascular plant lineage. On the other hand, particularly small group II introns and short intergenic regions reveal a tendency of evolution towards a compact mitochondrial genome. RNA editing reaches extreme levels exceeding 100 pyrimidine exchanges in individual mRNAs and, hitherto unobserved in such frequency, also in tRNAs with 18 C-to-U conversions in the tRNA for proline. In total, some 1500 sites of RNA editing can be expected for the Isoetes mitochondrial transcriptome. As a unique molecular novelty, the Isoetes cox1 gene requires trans-splicing via a discontinuous group I intron demonstrating disrupted, but functional, RNAs for yet another class of natural ribozymes.

Group II introns are large catalytic RNAs that are ancestrally related to nuclear spliceosomal introns. Sequences corresponding to group II RNAs are found in many prokaryotes and are particularly prevalent within plants organellar genomes. Proteins encoded within the introns themselves (maturases) facilitate the splicing of their own host pre-RNAs. Mitochondrial introns in plants have diverged considerably in sequence and have lost their maturases. In angiosperms, only a single maturase has been retained in the mitochondrial DNA: the matR gene found within NADH dehydrogenase 1 (nad1) intron 4. Its conservation across land plants and RNA editing events, which restore conserved amino acids, indicates that matR encodes a functional protein. However, the biological role of MatR remains unclear. Here, we performed an in vivo investigation of the roles of MatR in Brassicaceae. Directed knockdown of matR expression via synthetically designed ribozymes altered the processing of various introns, including nad1 i4. Pull-down experiments further indicated that MatR is associated with nad1 i4 and several other intron-containing pre-mRNAs. MatR may thus represent an intermediate link in the gradual evolutionary transition from the intron-specific maturases in bacteria into their versatile spliceosomal descendants in the nucleus. The similarity between maturases and the core spliceosomal Prp8 protein further supports this intriguing theory.

Most land plant plastomes contain two copies of a large inverted repeat (IR) that promote high-frequency homologous recombination to generate isomeric genomic forms. Among conifer plastomes, this canonical IR is highly reduced in Pinaceae and completely lost from cupressophytes. However, both lineages have acquired short, novel IRs, some of which also exhibit recombinational activity to generate genomic structural diversity. This diversity has been shown to exist between, and occasionally within, cupressophyte species, but it is not known whether multiple genomic forms coexist within individual plants. To examine the recombinational potential of the novel cupressophyte IRs within individuals and between species, we sequenced the plastomes of four closely related species of Juniperus. The four plastomes have identical gene content and genome organization except for a large 36 kb inversion between approximately 250 bp IR containing trnQ-UUG. Southern blotting showed that different isomeric versions of the plastome predominate among individual junipers, whereas polymerase chain reaction and high-throughput read-pair mapping revealed the substoichiometric presence of the alternative isomeric form within each individual plant. Furthermore, our comparative genomic studies demonstrate that the predominant and substoichiometric arrangements of this IR have changed several times in other cupressophytes as well. These results provide compelling evidence for substoichiometric shifting of plastomic forms during cupressophyte evolution and suggest that substoichiometric shifting activity in plastid genomes may be adaptive.

BackgroundPlastid genome structure and content is remarkably conserved in land plants. This widespread conservation has facilitated taxon-rich phylogenetic analyses that have resolved organismal relationships among many land plant groups. However, the relationships among major fern lineages, especially the placement of Equisetales, remain enigmatic.ResultsIn order to understand the evolution of plastid genomes and to establish phylogenetic relationships among ferns, we sequenced the plastid genomes from three early diverging species: Equisetum hyemale (Equisetales), Ophioglossum californicum (Ophioglossales), and Psilotum nudum (Psilotales). A comparison of fern plastid genomes showed that some lineages have retained inverted repeat (IR) boundaries originating from the common ancestor of land plants, while other lineages have experienced multiple IR changes including expansions and inversions. Genome content has remained stable throughout ferns, except for a few lineage-specific losses of genes and introns. Notably, the losses of the rps16 gene and the rps12i346 intron are shared among Psilotales, Ophioglossales, and Equisetales, while the gain of a mitochondrial atp1 intron is shared between Marattiales and Polypodiopsida. These genomic structural changes support the placement of Equisetales as sister to Ophioglossales + Psilotales and Marattiales as sister to Polypodiopsida. This result is augmented by some molecular phylogenetic analyses that recover the same relationships, whereas others suggest a relationship between Equisetales and Polypodiopsida.ConclusionsAlthough molecular analyses were inconsistent with respect to the position of Marattiales and Equisetales, several genomic structural changes have for the first time provided a clear placement of these lineages within the ferns. These results further demonstrate the power of using rare genomic structural changes in cases where molecular data fail to provide strong phylogenetic resolution.

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Summary The exchange of genetic material between cellular organelles through intracellular gene transfer (IGT) or between species by horizontal gene transfer (HGT) has played an important role in plant mitochondrial genome evolution. The mitochondrial genomes of Geraniaceae display a number of unusual phenomena including highly accelerated rates of synonymous substitutions, extensive gene loss and reduction in RNA editing. Mitochondrial DNA sequences assembled for 17 species of Geranium revealed substantial reduction in gene and intron content relative to the ancestor of the Geranium lineage. Comparative analyses of nuclear transcriptome data suggest that a number of these sequences have been functionally relocated to the nucleus via IGT. Evidence for rampant HGT was detected in several Geranium species containing foreign organellar DNA from diverse eudicots, including many transfers from parasitic plants. One lineage has experienced multiple, independent HGT episodes, many of which occurred within the past 5.5 Myr. Both duplicative and recapture HGT were documented in Geranium lineages. The mitochondrial genome of Geranium brycei contains at least four independent HGT tracts that are absent in its nearest relative. Furthermore, G. brycei mitochondria carry two copies of the cox1 gene that differ in intron content, providing insight into contrasting hypotheses on cox1 intron evolution.