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We sequenced mitochondrial genomes from five diverse diatoms (Toxarium undulatum, Psammoneis japonica, Eunotia naegelii, Cylindrotheca closterium, and Nitzschia sp.), chosen to fill important phylogenetic gaps and help us characterize broadscale patterns of mitochondrial genome evolution in diatoms. Although gene content was strongly conserved, intron content varied widely across species. The vast majority of introns were of group II type and were located in the cox1 or rnl genes. Although recurrent intron loss appears to be the principal underlying cause of the sporadic distributions of mitochondrial introns across diatoms, phylogenetic analyses showed that intron distributions superficially consistent with a recurrent-loss model were sometimes more complicated, implicating horizontal transfer as a likely mechanism of intron acquisition as well. It was not clear, however, whether diatoms were the donors or recipients of horizontally transferred introns, highlighting a general challenge in resolving the evolutionary histories of many diatom mitochondrial introns. Although some of these histories may become clearer as more genomes are sampled, high rates of intron loss suggest that the origins of many diatom mitochondrial introns are likely to remain unclear.

Background: The mitochondrial genomes of flowering plants vary greatly in size, gene content, gene order, mutation rate and level of RNA editing. However, the narrow phylogenetic breadth of available genomic data has limited our ability to reconstruct these traits in the ancestral flowering plant and, therefore, to infer subsequent patterns of evolution across angiosperms., Results: We sequenced the mitochondrial genome of Liriodendron tulipifera, the first from outside the monocots or eudicots. This 553,721 bp mitochondrial genome has evolved remarkably slowly in virtually all respects, with an extraordinarily low genome-wide silent substitution rate, retention of genes frequently lost in other angiosperm lineages, and conservation of ancestral gene clusters. The mitochondrial protein genes in Liriodendron are the most heavily edited of any angiosperm characterized to date. Most of these sites are also edited in various other lineages, which allowed us to polarize losses of editing sites in other parts of the angiosperm phylogeny. Finally, we added comprehensive gene sequence data for two other magnoliids, Magnolia stellata and the more distantly related Calycanthus floridus, to measure rates of sequence evolution in Liriodendron with greater accuracy. The Magnolia genome has evolved at an even lower rate, revealing a roughly 5,000-fold range of synonymous-site divergence among angiosperms whose mitochondrial gene space has been comprehensively sequenced., Conclusions: Using Liriodendron as a guide, we estimate that the ancestral flowering plant mitochondrial genome contained 41 protein genes, 14 tRNA genes of mitochondrial origin, as many as 7 tRNA genes of chloroplast origin, >700 sites of RNA editing, and some 14 colinear gene clusters. Many of these gene clusters, genes and RNA editing sites have been variously lost in different lineages over the course of the ensuing ∽200 million years of angiosperm evolution.

Genome size and complexity vary tremendously among eukaryotic species and their organelles. Comparisons across deeply divergent eukaryotic lineages have suggested that variation in mutation rates may explain this diversity, with increased mutational burdens favoring reduced genome size and complexity. The discovery that mitochondrial mutation rates can differ by orders of magnitude among closely related angiosperm species presents a unique opportunity to test this hypothesis. We sequenced the mitochondrial genomes from two species in the angiosperm genus Silene with recent and dramatic accelerations in their mitochondrial mutation rates. Contrary to theoretical predictions, these genomes have experienced a massive proliferation of noncoding content. At 6.7 and 11.3 Mb, they are by far the largest known mitochondrial genomes, larger than most bacterial genomes and even some nuclear genomes. In contrast, two slowly evolving Silene mitochondrial genomes are smaller than average for angiosperms. Consequently, this genus captures approximately 98% of known variation in organelle genome size. The expanded genomes reveal several architectural changes, including the evolution of complex multichromosomal structures (with 59 and 128 circular-mapping chromosomes, ranging in size from 44 to 192 kb). They also exhibit a substantial reduction in recombination and gene conversion activity as measured by the relative frequency of alternative genome conformations and the level of sequence divergence between repeat copies. The evolution of mutation rate, genome size, and chromosome structure can therefore be extremely rapid and interrelated in ways not predicted by current evolutionary theories. Our results raise the hypothesis that changes in recombinational processes, including gene conversion, may be a central force driving the evolution of both mutation rate and genome structure., Competing Interests: The authors have declared that no competing interests exist.

The angiosperm genus Silene exhibits some of the most extreme and rapid divergence ever identified in mitochondrial genome architecture and nucleotide substitution rates. These patterns have been considered mitochondrial specific based on the absence of correlated changes in the small number of available nuclear and plastid gene sequences. To better assess the relationship between mitochondrial and plastid evolution, we sequenced the plastid genomes from four Silene species with fully sequenced mitochondrial genomes. We found that two species with fast-evolving mitochondrial genomes, S. noctiflora and S. conica, also exhibit accelerated rates of sequence and structural evolution in their plastid genomes. The nature of these changes, however, is markedly different from those in the mitochondrial genome. For example, in contrast to the mitochondrial pattern, which appears to be genome wide and mutationally driven, the plastid substitution rate accelerations are restricted to a subset of genes and preferentially affect nonsynonymous sites, indicating that altered selection pressures are acting on specific plastid-encoded functions in these species. Indeed, some plastid genes in S. noctiflora and S. conica show strong evidence of positive selection. In contrast, two species with more slowly evolving mitochondrial genomes, S. latifolia and S. vulgaris, have correspondingly low rates of nucleotide substitution in plastid genes as well as a plastid genome structure that has remained essentially unchanged since the origin of angiosperms. These results raise the possibility that common evolutionary forces could be shaping the extreme but distinct patterns of divergence in both organelle genomes within this genus.

Members of the flowering plant family Cucurbitaceae harbor the largest known mitochondrial genomes. Here, we report the 1685-kb mitochondrial genome of cucumber (Cucumis sativus). We help solve a 30-year mystery about the origins of its large size by showing that it mainly reflects the proliferation of dispersed repeats, expansions of existing introns, and the acquisition of sequences from diverse sources, including the cucumber nuclear and chloroplast genomes, viruses, and bacteria. The cucumber genome has a novel structure for plant mitochondria, mapping as three entirely or largely autonomous circular chromosomes (lengths 1556, 84, and 45 kb) that vary in relative abundance over a twofold range. These properties suggest that the three chromosomes replicate independently of one another. The two smaller chromosomes are devoid of known functional genes but nonetheless contain diagnostic mitochondrial features. Paired-end sequencing conflicts reveal differences in recombination dynamics among chromosomes, for which an explanatory model is developed, as well as a large pool of low-frequency genome conformations, many of which may result from asymmetric recombination across intermediate-sized and sometimes highly divergent repeats. These findings highlight the promise of genome sequencing for elucidating the recombinational dynamics of plant mitochondrial genomes.

The mitochondrial genomes of seed plants are exceptionally fluid in size, structure, and sequence content, with the accumulation and activity of repetitive sequences underlying much of this variation. We report the first fully sequenced mitochondrial genome of a legume, Vigna radiata (mung bean), and show that despite its unexceptional size (401,262 nt), the genome is unusually depauperate in repetitive DNA and "promiscuous" sequences from the chloroplast and nuclear genomes. Although Vigna lacks the large, recombinationally active repeats typical of most other seed plants, a PCR survey of its modest repertoire of short (38-297 nt) repeats nevertheless revealed evidence for recombination across all of them. A set of novel control assays showed, however, that these results could instead reflect, in part or entirely, artifacts of PCR-mediated recombination. Consequently, we recommend that other methods, especially high-depth genome sequencing, be used instead of PCR to infer patterns of plant mitochondrial recombination. The average-sized but repeat- and feature-poor mitochondrial genome of Vigna makes it ever more difficult to generalize about the factors shaping the size and sequence content of plant mitochondrial genomes.

Background: Mitochondrial gene loss and functional transfer to the nucleus is an ongoing process in many lineages of plants, resulting in substantial variation across species in mitochondrial gene content. The Caryophyllaceae represents one lineage that has experienced a particularly high rate of mitochondrial gene loss relative to other angiosperms., Results: In this study, we report the first complete mitochondrial genome sequence from a member of this family, Silene latifolia. The genome can be mapped as a 253,413 bp circle, but its structure is complicated by a large repeated region that is present in 6 copies. Active recombination among these copies produces a suite of alternative genome configurations that appear to be at or near "recombinational equilibrium". The genome contains the fewest genes of any angiosperm mitochondrial genome sequenced to date, with intact copies of only 25 of the 41 protein genes inferred to be present in the common ancestor of angiosperms. As observed more broadly in angiosperms, ribosomal proteins have been especially prone to gene loss in the S. latifolia lineage. The genome has also experienced a major reduction in tRNA gene content, including loss of functional tRNAs of both native and chloroplast origin. Even assuming expanded wobble-pairing rules, the mitochondrial genome can support translation of only 17 of the 61 sense codons, which code for only 9 of the 20 amino acids. In addition, genes encoding 18S and, especially, 5S rRNA exhibit exceptional sequence divergence relative to other plants. Divergence in one region of 18S rRNA appears to be the result of a gene conversion event, in which recombination with a homologous gene of chloroplast origin led to the complete replacement of a helix in this ribosomal RNA., Conclusions: These findings suggest a markedly expanded role for nuclear gene products in the translation of mitochondrial genes in S. latifolia and raise the possibility of altered selective constraints operating on the mitochondrial translational apparatus in this lineage.

Theoretical arguments suggest that mutation rates influence the proliferation and maintenance of RNA editing. We identified RNA editing sites in five species within the angiosperm genus Silene that exhibit highly divergent mitochondrial mutation rates. We found that mutational acceleration has been associated with rapid loss of mitochondrial editing sites. In contrast, we did not find a significant difference in the frequency of editing in chloroplast genes, which lack the mutation rate variation observed in the mitochondrial genome. As found in other angiosperms, the rate of substitution at RNA editing sites in Silene greatly exceeds the rate at synonymous sites, a pattern that has previously been interpreted as evidence for selection against RNA editing. Alternatively, we suggest that editing sites may experience higher rates of C-to-T mutation than other portions of the genome. Such a pattern could be caused by gene conversion with reverse-transcribed mRNA (i.e., retroprocessing). If so, the genomic distribution of RNA editing site losses in Silene suggests that such conversions must be occurring at a local scale such that only one or two editing sites are affected at a time. Because preferential substitution at editing sites appears to occur in angiosperms regardless of the mutation rate, we conclude that mitochondrial rate accelerations within Silene have "fast-forwarded" a preexisting pattern but have not fundamentally changed the evolutionary forces acting on RNA editing sites.

The mitochondrial genomes of seed plants are unusually large and vary in size by at least an order of magnitude. Much of this variation occurs within a single family, the Cucurbitaceae, whose genomes range from an estimated 390 to 2,900 kb in size. We sequenced the mitochondrial genomes of Citrullus lanatus (watermelon: 379,236 nt) and Cucurbita pepo (zucchini: 982,833 nt)--the two smallest characterized cucurbit mitochondrial genomes--and determined their RNA editing content. The relatively compact Citrullus mitochondrial genome actually contains more and longer genes and introns, longer segmental duplications, and more discernibly nuclear-derived DNA. The large size of the Cucurbita mitochondrial genome reflects the accumulation of unprecedented amounts of both chloroplast sequences (>113 kb) and short repeated sequences (>370 kb). A low mutation rate has been hypothesized to underlie increases in both genome size and RNA editing frequency in plant mitochondria. However, despite its much larger genome, Cucurbita has a significantly higher synonymous substitution rate (and presumably mutation rate) than Citrullus but comparable levels of RNA editing. The evolution of mutation rate, genome size, and RNA editing are apparently decoupled in Cucurbitaceae, reflecting either simple stochastic variation or governance by different factors.