Novel genetic code and record-setting AT-richness in the highly reduced plastid genome of the holoparasitic plant Balanophora.

Plastid genomes (plastomes) vary enormously in size and gene content among the many lineages of nonphotosynthetic plants, but key lineages remain unexplored. We therefore investigated plastome sequence and expression in the holoparasitic and morphologically bizarre Balanophoraceae. The two Balanophora plastomes examined are remarkable, exhibiting features rarely if ever seen before in plastomes or in any other genomes. At 15.5 kb in size and with only 19 genes, they are among the most reduced plastomes known. They have no tRNA genes for protein synthesis, a trait found in only three other plastid lineages, and thus Balanophora plastids must import all tRNAs needed for translation. Balanophora plastomes are exceptionally compact, with numerous overlapping genes, highly reduced spacers, loss of all cis-spliced introns, and shrunken protein genes. With A+T contents of 87.8% and 88.4%, the Balanophora genomes are the most AT-rich genomes known save for a single mitochondrial genome that is merely bloated with AT-rich spacer DNA. Most plastid protein genes in Balanophora consist of ≥90% AT, with several between 95% and 98% AT, resulting in the most biased codon usage in any genome described to date. A potential consequence of its radical compositional evolution is the novel genetic code used by Balanophora plastids, in which TAG has been reassigned from stop to tryptophan. Despite its many exceptional properties, the Balanophora plastome must be functional because all examined genes are transcribed, its only intron is correctly trans-spliced, and its protein genes, although highly divergent, are evolving under various degrees of selective constraint.

Horizontal gene transfers dominate the functional mitochondrial gene space of a holoparasitic plant.

Although horizontal gene transfer (HGT) is common in angiosperm mitochondrial DNAs (mtDNAs), few cases of functional foreign genes have been identified. The one outstanding candidate for large-scale functional HGT is the holoparasite Lophophytum mirabile, whose mtDNA has lost most native genes but contains intact foreign homologs acquired from legume host plants. To investigate the extent to which this situation results from functional replacement of native by foreign genes, functional mitochondrial gene transfer to the nucleus, and/or loss of mitochondrial biochemical function in the context of extreme parasitism, we examined the Lophophytum mitochondrial and nuclear transcriptomes by deep paired-end RNA sequencing. Most foreign mitochondrial genes in Lophophytum are highly transcribed, accurately spliced, and efficiently RNA edited. By contrast, we found no evidence for functional gene transfer to the nucleus or loss of mitochondrial functions in Lophophytum. Many functional replacements occurred via the physical replacement of native genes by foreign genes. Some of these events probably occurred as the final act of HGT itself. Lophophytum mtDNA has experienced an unprecedented level of functional replacement of native genes by foreign copies. This raises important questions concerning population-genetic and molecular regimes that underlie such a high level of foreign gene takeover.

Organellomic data sets confirm a cryptic consensus on (unrooted) land-plant relationships and provide new insights into bryophyte molecular evolution.

PREMISE: Phylogenetic trees of bryophytes provide important evolutionary context for land plants. However, published inferences of overall embryophyte relationships vary considerably. We performed phylogenomic analyses of bryophytes and relatives using both mitochondrial and plastid gene sets, and investigated bryophyte plastome evolution. METHODS: We employed diverse likelihood-based analyses to infer large-scale bryophyte phylogeny for mitochondrial and plastid data sets. We tested for changes in purifying selection in plastid genes of a mycoheterotrophic liverwort (Aneura mirabilis) and a putatively mycoheterotrophic moss (Buxbaumia), and compared 15 bryophyte plastomes for major structural rearrangements. RESULTS: Overall land-plant relationships conflict across analyses, generally weakly. However, an underlying (unrooted) four-taxon tree is consistent across most analyses and published studies. Despite gene coverage patchiness, relationships within mosses, liverworts, and hornworts are largely congruent with previous studies, with plastid results generally better supported. Exclusion of RNA edit sites restores cases of unexpected non-monophyly to monophyly for Takakia and two hornwort genera. Relaxed purifying selection affects multiple plastid genes in mycoheterotrophic Aneura but not Buxbaumia. Plastid genome structure is nearly invariant across bryophytes, but the tufA locus, presumed lost in embryophytes, is unexpectedly retained in several mosses. CONCLUSIONS: A common unrooted tree underlies embryophyte phylogeny, [(liverworts, mosses), (hornworts, vascular plants)]; rooting inconsistency across studies likely reflects substantial distance to algal outgroups. Analyses combining genomic and transcriptomic data may be misled locally for heavily RNA-edited taxa. The Buxbaumia plastome lacks hallmarks of relaxed selection found in mycoheterotrophic Aneura. Autotrophic bryophyte plastomes, including Buxbaumia, hardly vary in overall structure.

High and Variable Rates of Repeat-Mediated Mitochondrial Genome Rearrangement in a Genus of Plants.

For 30 years, it has been clear that angiosperm mitochondrial genomes evolve rapidly in sequence arrangement (i.e., synteny), yet absolute rates of rearrangement have not been measured in any plant group, nor is it known how much these rates vary. To investigate these issues, we sequenced and reconstructed the rearrangement history of seven mitochondrial genomes in Monsonia (Geraniaceae). We show that rearrangements (occurring mostly as inversions) not only take place at generally high rates in these genomes but also uncover significant variation in rearrangement rates. For example, the hyperactive mitochondrial genome of Monsonia ciliata has accumulated at least 30 rearrangements over the last million years, whereas the branch leading to M. ciliata and its sister species has sustained rearrangement at a rate that is at least ten times lower. Furthermore, our analysis of published data shows that rates of mitochondrial genome rearrangement in seed plants vary by at least 600-fold. We find that sites of rearrangement are highly preferentially located in very close proximity to repeated sequences in Monsonia. This provides strong support for the hypothesis that rearrangement in angiosperm mitochondrial genomes occurs largely through repeat-mediated recombination. Because there is little variation in the amount of repeat sequence among Monsonia genomes, the variable rates of rearrangement in Monsonia probably reflect variable rates of mitochondrial recombination itself. Finally, we show that mitochondrial synonymous substitutions occur in a clock-like manner in Monsonia; rates of mitochondrial substitutions and rearrangements are therefore highly uncoupled in this group.

Mitochondrial Retroprocessing Promoted Functional Transfers of rpl5 to the Nucleus in Grasses.

Functional gene transfers from the mitochondrion to the nucleus are ongoing in angiosperms and have occurred repeatedly for all 15 ribosomal protein genes, but it is not clear why some of these genes are transferred more often than others nor what the balance is between DNA- and RNA-mediated transfers. Although direct insertion of mitochondrial DNA into the nucleus occurs frequently in angiosperms, case studies of functional mitochondrial gene transfer have implicated an RNA-mediated mechanism that eliminates introns and RNA editing sites, which would otherwise impede proper expression of mitochondrial genes in the nucleus. To elucidate the mechanisms that facilitate functional gene transfers and the evolutionary dynamics of the coexisting nuclear and mitochondrial gene copies that are established during these transfers, we have analyzed rpl5 genes from 90 grasses (Poaceae) and related monocots. Multiple lines of evidence indicate that rpl5 has been functionally transferred to the nucleus at least three separate times in the grass family and that at least seven species have intact and transcribed (but not necessarily functional) copies in both the mitochondrion and nucleus. In two grasses, likely functional nuclear copies of rpl5 have been subject to recent gene conversion events via secondarily transferred mitochondrial copies in what we believe are the first described cases of mitochondrial-to-nuclear gene conversion. We show that rpl5 underwent a retroprocessing event within the mitochondrial genome early in the evolution of the grass family, which we argue predisposed the gene towards successful, DNA-mediated functional transfer by generating a "pre-edited" sequence.

Miniaturized mitogenome of the parasitic plant Viscum scurruloideum is extremely divergent and dynamic and has lost all nad genes.

Despite the enormous diversity among parasitic angiosperms in form and structure, life-history strategies, and plastid genomes, little is known about the diversity of their mitogenomes. We report the sequence of the wonderfully bizarre mitogenome of the hemiparasitic aerial mistletoe Viscum scurruloideum. This genome is only 66 kb in size, making it the smallest known angiosperm mitogenome by a factor of more than three and the smallest land plant mitogenome. Accompanying this size reduction is exceptional reduction of gene content. Much of this reduction arises from the unexpected loss of respiratory complex I (NADH dehydrogenase), universally present in all 300+ other angiosperms examined, where it is encoded by nine mitochondrial and many nuclear nad genes. Loss of complex I in a multicellular organism is unprecedented. We explore the potential relationship between this loss in Viscum and its parasitic lifestyle. Despite its small size, the Viscum mitogenome is unusually rich in recombinationally active repeats, possessing unparalleled levels of predicted sublimons resulting from recombination across short repeats. Many mitochondrial gene products exhibit extraordinary levels of divergence in Viscum, indicative of highly relaxed if not positive selection. In addition, all Viscum mitochondrial protein genes have experienced a dramatic acceleration in synonymous substitution rates, consistent with the hypothesis of genomic streamlining in response to a high mutation rate but completely opposite to the pattern seen for the high-rate but enormous mitogenomes of Silene. In sum, the Viscum mitogenome possesses a unique constellation of extremely unusual features, a subset of which may be related to its parasitic lifestyle.

Ginkgo and Welwitschia Mitogenomes Reveal Extreme Contrasts in Gymnosperm Mitochondrial Evolution.

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.

Comparative mitogenomics indicates respiratory competence in parasitic Viscum despite loss of complex I and extreme sequence divergence, and reveals horizontal gene transfer and remarkable variation in genome size.

BACKGROUND: Aerobically respiring eukaryotes usually contain four respiratory-chain complexes (complexes I-IV) and an ATP synthase (complex V). In several lineages of aerobic microbial eukaryotes, complex I has been lost, with an alternative, nuclear-encoded NADH dehydrogenase shown in certain cases to bypass complex I and oxidize NADH without proton translocation. The first loss of complex I in any multicellular eukaryote was recently reported in two studies; one sequenced the complete mitogenome of the hemiparasitic aerial mistletoe, Viscum scurruloideum, and the other sequenced the V. album mitogenome. The V. scurruloideum study reported no significant additional loss of mitochondrial genes or genetic function, but the V. album study postulated that mitochondrial genes encoding all ribosomal RNAs and proteins of all respiratory complexes are either absent or pseudogenes, thus raising questions as to whether the mitogenome and oxidative respiration are functional in this plant. RESULTS: To determine whether these opposing conclusions about the two Viscum mitogenomes reflect a greater degree of reductive/degenerative evolution in V. album or instead result from interpretative and analytical differences, we reannotated and reanalyzed the V. album mitogenome and compared it with the V. scurruloideum mitogenome. We find that the two genomes share a complete complement of mitochondrial rRNA genes and a typical complement of genes encoding respiratory complexes II-V. Most Viscum mitochondrial protein genes exhibit very high levels of divergence yet are evolving under purifying, albeit relaxed selection. We discover two cases of horizontal gene transfer in V. album and show that the two Viscum mitogenomes differ by 8.6-fold in size (66 kb in V. scurruloideum; 565 kb in V. album). CONCLUSIONS: Viscum mitogenomes are extraordinary compared to other plant mitogenomes in terms of their wide size range, high rates of synonymous substitutions, degree of relaxed selection, and unprecedented loss of respiratory complex I. However, contrary to the initial conclusions regarding V. album, both Viscum mitogenomes possess conventional sets of rRNA and, excepting complex I, respiratory genes. Both plants should therefore be able to carry out aerobic respiration. Moreover, with respect to size, the V. scurruloideum mitogenome has experienced a greater level of reductive evolution.

Unique role for translation initiation factor 3 in the light color regulation of photosynthetic gene expression.

Light-harvesting antennae are critical for collecting energy from sunlight and providing it to photosynthetic reaction centers. Their abundance and composition are tightly regulated to maintain efficient photosynthesis in changing light conditions. Many cyanobacteria alter their light-harvesting antennae in response to changes in ambient light-color conditions through the process of chromatic acclimation. The control of green light induction (Cgi) pathway is a light-color-sensing system that controls the expression of photosynthetic genes during chromatic acclimation, and while some evidence suggests that it operates via transcription attenuation, the components of this pathway have not been identified. We provide evidence that translation initiation factor 3 (IF3), an essential component of the prokaryotic translation initiation machinery that binds the 30S subunit and blocks premature association with the 50S subunit, is part of the control of green light induction pathway. Light regulation of gene expression has not been previously described for any translation initiation factor. Surprisingly, deletion of the IF3-encoding gene infCa was not lethal in the filamentous cyanobacterium Fremyella diplosiphon, and its genome was found to contain a second, redundant, highly divergent infC gene which, when deleted, had no effect on photosynthetic gene expression. Either gene could complement an Escherichia coli infC mutant and thus both encode bona fide IF3s. Analysis of prokaryotic and eukaryotic genome databases established that multiple infC genes are present in the genomes of diverse groups of bacteria and land plants, most of which do not undergo chromatic acclimation. This suggests that IF3 may have repeatedly evolved important roles in the regulation of gene expression in both prokaryotes and eukaryotes.

Homologous recombination and retention of a single form of most genes shape the highly chimeric mitochondrial genome of a cybrid plant.

The structure and evolution of angiosperm mitochondrial genomes are driven by extremely high rates of recombination and rearrangement. An excellent experimental system for studying these events is offered by cybrid plants, in which parental mitochondria usually fuse and their genomes recombine. Little is known about the extent, nature and consequences of mitochondrial recombination in these plants. We conducted the first study in which the organellar genomes of a cybrid - between Nicotiana tabacum and Hyoscyamus niger - were sequenced and compared to those of its parents. This cybrid mitochondrial genome is highly recombinant, reflecting at least 30 crossovers and five gene conversions between its parental genomes. It is also surprisingly large (41% and 64% larger than the parental genomes), yet contains single alleles for 90% of mitochondrial genes. Recombination produced a remarkably chimeric cybrid mitochondrial genome and occurred entirely via homologous mechanisms involving the double-strand break repair and/or break-induced replication pathways. Retention of a single form of most genes could be advantageous to minimize intracellular incompatibilities and/or reflect neutral forces that preferentially eliminate duplicated regions. We discuss the relevance of these findings to the surprisingly frequent occurrence of horizontal gene - and genome - transfer in angiosperm mitochondrial DNAs.

Rapid evolution of enormous, multichromosomal genomes in flowering plant mitochondria with exceptionally high mutation rates.

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.

Origins and recombination of the bacterial-sized multichromosomal mitochondrial genome of cucumber.

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.

Horizontal gene transfer in eukaryotic evolution.

Horizontal gene transfer (HGT; also known as lateral gene transfer) has had an important role in eukaryotic genome evolution, but its importance is often overshadowed by the greater prevalence and our more advanced understanding of gene transfer in prokaryotes. Recurrent endosymbioses and the generally poor sampling of most nuclear genes from diverse lineages have also complicated the search for transferred genes. Nevertheless, the number of well-supported cases of transfer from both prokaryotes and eukaryotes, many with significant functional implications, is now expanding rapidly. Major recent trends include the important role of HGT in adaptation to certain specialized niches and the highly variable impact of HGT in different lineages.

The "fossilized" mitochondrial genome of Liriodendron tulipifera: ancestral gene content and order, ancestral editing sites, and extraordinarily low mutation rate.

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.

Localized hypermutation and associated gene losses in legume chloroplast genomes.

Point mutations result from errors made during DNA replication or repair, so they are usually expected to be homogeneous across all regions of a genome. However, we have found a region of chloroplast DNA in plants related to sweetpea (Lathyrus) whose local point mutation rate is at least 20 times higher than elsewhere in the same molecule. There are very few precedents for such heterogeneity in any genome, and we suspect that the hypermutable region may be subject to an unusual process such as repeated DNA breakage and repair. The region is 1.5 kb long and coincides with a gene, ycf4, whose rate of evolution has increased dramatically. The product of ycf4, a photosystem I assembly protein, is more divergent within the single genus Lathyrus than between cyanobacteria and other angiosperms. Moreover, ycf4 has been lost from the chloroplast genome in Lathyrus odoratus and separately in three other groups of legumes. Each of the four consecutive genes ycf4-psaI-accD-rps16 has been lost in at least one member of the legume "inverted repeat loss" clade, despite the rarity of chloroplast gene losses in angiosperms. We established that accD has relocated to the nucleus in Trifolium species, but were unable to find nuclear copies of ycf4 or psaI in Lathyrus. Our results suggest that, as well as accelerating sequence evolution, localized hypermutation has contributed to the phenomenon of gene loss or relocation to the nucleus.

Gorgeous mosaic of mitochondrial genes created by horizontal transfer and gene conversion.

The best known outcome of horizontal gene transfer (HGT) is the introduction of novel genes, but other outcomes have been described. When a transferred gene has a homolog in the recipient genome, the native gene may be functionally replaced (and subsequently lost) or partially overwritten by gene conversion with transiently present foreign DNA. Here we report the discovery, in two lineages of plant mitochondrial genes, of novel gene combinations that arose by conversion between coresident native and foreign homologs. These lineages have undergone intricate conversion between native and foreign copies, with conversion occurring repeatedly and differentially over the course of speciation, leading to radiations of mosaic genes involved in respiration and intron splicing. Based on these findings, we develop a model--the duplicative HGT and differential gene conversion model--that integrates HGT and ongoing gene conversion in the context of speciation. Finally, we show that one of these HGT-driven gene-conversional radiations followed two additional types of conversional chimerism, namely, intramitochondrial retroprocessing and interorganellar gene conversion across the 2 billion year divide between mitochondria and chloroplasts. These findings expand our appreciation of HGT and gene conversion as creative evolutionary forces, establish plant mitochondria as a premiere system for studying the evolutionary dynamics of HGT and its genetic reverberations, and recommend careful examination of bacterial and other genomes for similar, likely overlooked phenomena.

Fine-scale mergers of chloroplast and mitochondrial genes create functional, transcompartmentally chimeric mitochondrial genes.

The mitochondrial genomes of flowering plants possess a promiscuous proclivity for taking up sequences from the chloroplast genome. All characterized chloroplast integrants exist apart from native mitochondrial genes, and only a few, involving chloroplast tRNA genes that have functionally supplanted their mitochondrial counterparts, appear to be of functional consequence. We developed a novel computational approach to search for homologous recombination (gene conversion) in a large number of sequences and applied it to 22 mitochondrial and chloroplast gene pairs, which last shared common ancestry some 2 billion years ago. We found evidence of recurrent conversion of short patches of mitochondrial genes by chloroplast homologs during angiosperm evolution, but no evidence of gene conversion in the opposite direction. All 9 putative conversion events involve the atp1/atpA gene encoding the alpha subunit of ATP synthase, which is unusually well conserved between the 2 organelles and the only shared gene that is widely sequenced across plant mitochondria. Moreover, all conversions were limited to the 2 regions of greatest nucleotide and amino acid conservation of atp1/atpA. These observations probably reflect constraints operating on both the occurrence and fixation of recombination between ancient homologs. These findings indicate that recombination between anciently related sequences is more frequent than previously appreciated and creates functional mitochondrial genes of chimeric origin. These results also have implications for the widespread use of mitochondrial atp1 in phylogeny reconstruction.

Multiple recent horizontal transfers of the cox1 intron in Solanaceae and extended co-conversion of flanking exons.

BACKGROUND: The most frequent case of horizontal transfer in plants involves a group I intron in the mitochondrial gene cox1, which has been acquired via some 80 separate plant-to-plant transfer events among 833 diverse angiosperms examined. This homing intron encodes an endonuclease thought to promote the intron's promiscuous behavior. A promising experimental approach to study endonuclease activity and intron transmission involves somatic cell hybridization, which in plants leads to mitochondrial fusion and genome recombination. However, the cox1 intron has not yet been found in the ideal group for plant somatic genetics - the Solanaceae. We therefore undertook an extensive survey of this family to find members with the intron and to learn more about the evolutionary history of this exceptionally mobile genetic element. RESULTS: Although 409 of the 426 species of Solanaceae examined lack the cox1 intron, it is uniformly present in three phylogenetically disjunct clades. Despite strong overall incongruence of cox1 intron phylogeny with angiosperm phylogeny, two of these clades possess nearly identical intron sequences and are monophyletic in intron phylogeny. These two clades, and possibly the third also, contain a co-conversion tract (CCT) downstream of the intron that is extended relative to all previously recognized CCTs in angiosperm cox1. Re-examination of all published cox1 genes uncovered additional cases of extended co-conversion and identified a rare case of putative intron loss, accompanied by full retention of the CCT. CONCLUSIONS: We infer that the cox1 intron was separately and recently acquired by at least three different lineages of Solanaceae. The striking identity of the intron and CCT from two of these lineages suggests that one of these three intron captures may have occurred by a within-family transfer event. This is consistent with previous evidence that horizontal transfer in plants is biased towards phylogenetically local events. The discovery of extended co-conversion suggests that other cox1 conversions may be longer than realized but obscured by the exceptional conservation of plant mitochondrial sequences. Our findings provide further support for the rampant-transfer model of cox1 intron evolution and recommend the Solanaceae as a model system for the experimental analysis of cox1 intron transfer in plants.

Insights into the evolution of mitochondrial genome size from complete sequences of Citrullus lanatus and Cucurbita pepo (Cucurbitaceae).

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.

Horizontal acquisition of multiple mitochondrial genes from a parasitic plant followed by gene conversion with host mitochondrial genes.

BACKGROUND: Horizontal gene transfer (HGT) is relatively common in plant mitochondrial genomes but the mechanisms, extent and consequences of transfer remain largely unknown. Previous results indicate that parasitic plants are often involved as either transfer donors or recipients, suggesting that direct contact between parasite and host facilitates genetic transfer among plants. RESULTS: In order to uncover the mechanistic details of plant-to-plant HGT, the extent and evolutionary fate of transfer was investigated between two groups: the parasitic genus Cuscuta and a small clade of Plantago species. A broad polymerase chain reaction (PCR) survey of mitochondrial genes revealed that at least three genes (atp1, atp6 and matR) were recently transferred from Cuscuta to Plantago. Quantitative PCR assays show that these three genes have a mitochondrial location in the one species line of Plantago examined. Patterns of sequence evolution suggest that these foreign genes degraded into pseudogenes shortly after transfer and reverse transcription (RT)-PCR analyses demonstrate that none are detectably transcribed. Three cases of gene conversion were detected between native and foreign copies of the atp1 gene. The identical phylogenetic distribution of the three foreign genes within Plantago and the retention of cytidines at ancestral positions of RNA editing indicate that these genes were probably acquired via a single, DNA-mediated transfer event. However, samplings of multiple individuals from two of the three species in the recipient Plantago clade revealed complex and perplexing phylogenetic discrepancies and patterns of sequence divergence for all three of the foreign genes. CONCLUSIONS: This study reports the best evidence to date that multiple mitochondrial genes can be transferred via a single HGT event and that transfer occurred via a strictly DNA-level intermediate. The discovery of gene conversion between co-resident foreign and native mitochondrial copies suggests that transferred genes may be evolutionarily important in generating mitochondrial genetic diversity. Finally, the complex relationships within each lineage of transferred genes imply a surprisingly complicated history of these genes in Plantago subsequent to their acquisition via HGT and this history probably involves some combination of additional transfers (including intracellular transfer), gene duplication, differential loss and mutation-rate variation. Unravelling this history will probably require sequencing multiple mitochondrial and nuclear genomes from Plantago. See Commentary: http://www.biomedcentral.com/1741-7007/8/147.