The mitochondrial genome of Malus domestica and the import-driven hypothesis of mitochondrial genome expansion in seed plants

Mitochondrial genomes of spermatophytes are the largest of all organellar genomes. Their large size has been attributed to various factors; however, the relative contribution of these factors to mitochondrial DNA (mtDNA) expansion remains undetermined. We estimated their relative contribution in Malus domestica (apple). The mitochondrial genome of apple has a size of 396 947 bp and a one to nine ratio of coding to non-coding DNA, close to the corresponding average values for angiosperms. We determined that 71.5% of the apple mtDNA sequence was highly similar to sequences of its nuclear DNA. Using nuclear gene exons, nuclear transposable elements and chloroplast DNA as markers of promiscuous DNA content in mtDNA, we estimated that approximately 20% of the apple mtDNA consisted of DNA sequences imported from other cell compartments, mostly from the nucleus. Similar marker-based estimates of promiscuous DNA content in the mitochondrial genomes of other species ranged between 21.2 and 25.3% of the total mtDNA length for grape, between 23.1 and 38.6% for rice, and between 47.1 and 78.4% for maize. All these estimates are conservative, because they underestimate the import of non-functional DNA. We propose that the import of promiscuous DNA is a core mechanism for mtDNA size expansion in seed plants. In apple, maize and grape this mechanism contributed far more to genome expansion than did homologous recombination. In rice the estimated contribution of both mechanisms was found to be similar.     

Rapid sequencing of the bamboo mitochondrial genome using Illumina technology and parallel episodic evolution of organelle genomes in grasses

Background

Compared to their counterparts in animals, the mitochondrial (mt) genomes of angiosperms exhibit a number of unique features. However, unravelling their evolution is hindered by the few completed genomes, of which are essentially Sanger sequenced. While next-generation sequencing technologies have revolutionized chloroplast genome sequencing, they are just beginning to be applied to angiosperm mt genomes. Chloroplast genomes of grasses (Poaceae) have undergone episodic evolution and the evolutionary rate was suggested to be correlated between chloroplast and mt genomes in Poaceae. It is interesting to investigate whether correlated rate change also occurred in grass mt genomes as expected under lineage effects. A time-calibrated phylogenetic tree is needed to examine rate change.

Methodology/Principal Findings

We determined a largely completed mt genome from a bamboo, Ferrocalamus rimosivaginus (Poaceae), through Illumina sequencing of total DNA. With combination of de novo and reference-guided assembly, 39.5-fold coverage Illumina reads were finally assembled into scaffolds totalling 432,839 bp. The assembled genome contains nearly the same genes as the completed mt genomes in Poaceae. For examining evolutionary rate in grass mt genomes, we reconstructed a phylogenetic tree including 22 taxa based on 31 mt genes. The topology of the well-resolved tree was almost identical to that inferred from chloroplast genome with only minor difference. The inconsistency possibly derived from long branch attraction in mtDNA tree. By calculating absolute substitution rates, we found significant rate change (∼4-fold) in mt genome before and after the diversification of Poaceae both in synonymous and nonsynonymous terms. Furthermore, the rate change was correlated with that of chloroplast genomes in grasses.

Conclusions/Significance

Our result demonstrates that it is a rapid and efficient approach to obtain angiosperm mt genome sequences using Illumina sequencing technology. The parallel episodic evolution of mt and chloroplast genomes in grasses is consistent with lineage effects.     

Complete Nucleotide Sequence of the Chloroplast Genome from the Tasmanian Blue Gum, Eucalyptus globulus (Myrtaceae)

The complete nucleotide sequence of the chloroplast genome ofthe hardwood species Eucalyptus globulus is presented and comparedwith chloroplast genomes of tree and non-tree angiosperms andtwo softwood tree species. The 160 286 bp genome is similarin gene order to that of Nicotiana, with an inverted repeat(IR) (26 393 bp) separated by a large single copy (LSC) regionof 89 012 bp and a small single copy region of 18 488 bp. Thereare 128 genes (112 individual gene species and 16 genes duplicatedin the inverted repeat) coding for 30 transfer RNAs, 4 ribosomalRNAs and 78 proteins. One pseudogene (-infA) and one pseudo-ycf(-ycf15) were identified. The chloroplast genome of E. globulusis essentially co-linear with that of another hardwood treespecies, Populus trichocarpa, except that the latter lacks rps16and rpl32, and the IR has expanded in Populus to include rps19(part of the LSC in E. globulus). Since the chloroplast genomeof E. globulus is not significantly different from other treeand non-tree angiosperm taxa, a comparison of hardwood and softwoodchloroplasts becomes, in essence, a comparison of angiospermand gymnosperm chloroplasts. When compared with E. globulus,Pinus chloroplasts have a very small IR, two extra tRNAs andfour additional photosynthetic genes, lack any functional ndhgenes and have a significantly different genome arrangement.There does not appear to be any correlation between plant habitand chloroplast genome composition and arrangement.     

The complete chloroplast and mitochondrial genome sequences of Boea hygrometrica: insights into the evolution of plant organellar genomes

The complete nucleotide sequences of the chloroplast (cp) and mitochondrial (mt) genomes of resurrection plant Boea hygrometrica (Bh, Gesneriaceae) have been determined with the lengths of 153,493 bp and 510,519 bp, respectively. The smaller chloroplast genome contains more genes (147) with a 72% coding sequence, and the larger mitochondrial genome have less genes (65) with a coding faction of 12%. Similar to other seed plants, the Bh cp genome has a typical quadripartite organization with a conserved gene in each region. The Bh mt genome has three recombinant sequence repeats of 222 bp, 843 bp, and 1474 bp in length, which divide the genome into a single master circle (MC) and four isomeric molecules. Compared to other angiosperms, one remarkable feature of the Bh mt genome is the frequent transfer of genetic material from the cp genome during recent Bh evolution. We also analyzed organellar genome evolution in general regarding genome features as well as compositional dynamics of sequence and gene structure/organization, providing clues for the understanding of the evolution of organellar genomes in plants. The cp-derived sequences including tRNAs found in angiosperm mt genomes support the conclusion that frequent gene transfer events may have begun early in the land plant lineage.     

Free from mitochondrial DNA: Nuclear genes and the inference of species trees among closely related darter lineages (Teleostei: Percidae: Etheostomatinae)

Investigations into the phylogenetics of closely related animal species are dominated by the use of mitochondrial DNA (mtDNA) sequence data. However, the near-ubiquitous use of mtDNA to infer phylogeny among closely related animal lineages is tempered by an increasing number of studies that document high rates of transfer of mtDNA genomes among closely related species through hybridization, leading to substantial discordance between phylogenies inferred from mtDNA and nuclear gene sequences. In addition, the recent development of methods that simultaneously infer a species phylogeny and estimate divergence times, while accounting for incongruence among individual gene trees, has ushered in a new era in the investigation of phylogeny among closely related species. In this study we assess if DNA sequence data sampled from a modest number of nuclear genes can resolve relationships of a species-rich clade of North American freshwater teleost fishes, the darters. We articulate and expand on a recently introduced method to infer a time-calibrated multi-species coalescent phylogeny using the computer program ^*BEAST. Our analyses result in well-resolved and strongly supported time-calibrated darter species tree. Contrary to the expectation that mtDNA will provide greater phylogenetic resolution than nuclear gene data; the darter species tree inferred exclusively from nuclear genes exhibits a higher frequency of strongly supported nodes than the mtDNA time-calibrated gene tree.     

The complete chloroplast genome of Gracilariopsis lemaneiformis (Rhodophyta) gives new insight into the evolution of family Gracilariaceae

The complete chloroplast genome of Gracilariopsis lemaneiformis was recovered from a Next Generation Sequencing data set. Without quadripartite structure, this chloroplast genome (183,013 bp, 27.40% GC content) contains 202 protein‐coding genes, 34 tRNA genes, 3 rRNA genes, and 1 tmRNA gene. Synteny analysis showed plasmid incorporation regions in chloroplast genomes of three species of family Gracilariaceae and in Grateloupia taiwanensis of family Halymeniaceae. Combined with reported red algal plasmid sequences in nuclear and mitochondrial genomes, we postulated that red algal plasmids may have played an important role in ancient horizontal gene transfer among nuclear, chloroplast, and mitochondrial genomes. Substitution rate analysis showed that purifying selective forces maintaining stability of protein‐coding genes of nine red algal chloroplast genomes over long periods must be strong and that the forces acting on gene groups and single genes of nine red algal chloroplast genomes were similar and consistent. The divergence of Gp. lemaneiformis occurred ~447.98 million years ago (Mya), close to the divergence time of genus Pyropia and Porphyra (443.62 Mya).     

A physical map for the Amborella trichopoda genome sheds light on the evolution of angiosperm genome structure

Background

Recent phylogenetic analyses have identified Amborella trichopoda, an understory tree species endemic to the forests of New Caledonia, as sister to a clade including all other known flowering plant species. The Amborella genome is a unique reference for understanding the evolution of angiosperm genomes because it can serve as an outgroup to root comparative analyses. A physical map, BAC end sequences and sample shotgun sequences provide a first view of the 870 Mbp Amborella genome.

Results

Analysis of Amborella BAC ends sequenced from each contig suggests that the density of long terminal repeat retrotransposons is negatively correlated with that of protein coding genes. Syntenic, presumably ancestral, gene blocks were identified in comparisons of the Amborella BAC contigs and the sequenced Arabidopsis thaliana, Populus trichocarpa, Vitis vinifera and Oryza sativa genomes. Parsimony mapping of the loss of synteny corroborates previous analyses suggesting that the rate of structural change has been more rapid on lineages leading to Arabidopsis and Oryza compared with lineages leading to Populus and Vitis. The gamma paleohexiploidy event identified in the Arabidopsis, Populus and Vitis genomes is shown to have occurred after the divergence of all other known angiosperms from the lineage leading to Amborella.

Conclusions

When placed in the context of a physical map, BAC end sequences representing just 5.4% of the Amborella genome have facilitated reconstruction of gene blocks that existed in the last common ancestor of all flowering plants. The Amborella genome is an invaluable reference for inferences concerning the ancestral angiosperm and subsequent genome evolution.     

Analysis of organellar genomes in brown algae reveals an independent introduction of similar foreign sequences into the mitochondrial genome

Kelp species (Laminariales, Phaeophyceae) are globally widespread along temperate to Polar rocky coastal lines. Here we analyse the mitochondrial and chloroplast genomes of Laminaria rodriguezii, in comparison to the organellar genomes of other kelp species. We also provide the complete mitochondrial genome sequence of another endemic kelp species from a Polar habitat, the Arctic Laminaria solidungula. We compare phylogenetic trees derived from twenty complete mitochondrial and seven complete chloroplast kelp genomes. Interestingly, we found a stretch of more than 700 bp in the mitochondrial genome of L.rodriguezii, which is not present in any other yet sequenced member of the Phaeophyceae. This stretch matches a protein coding region in the mitochondrial genome from Desmarestia viridis, another brown seaweed. Their high similarity suggests that these sequences originated through independent introduction into the two species. Their origin could have been by infection by yet unknown similar mitoviruses, currently only known from fungi and plants.     

Identification of the entire set of transferred chloroplast DNA sequences in the mitochondrial genome of rice

Summary The entire set of transferred chloroplast DNA sequences in the mitochondrial genome of rice (Oryza sativa cv. Nipponbare) was identified using clone banks that cover the chloroplast and mitochondrial genomes. The mitochondrial fragments that were homologous to chloroplast DNA were mapped and sequenced. The nucleotide sequences around the termini of integrated chloroplast sequences in the rice mtDNA revealed no common sequences or structures that might enhance the transfer of DNA. Sixteen chloroplast sequences, ranging from 32 bases to 6.8 kb in length, were found to be dispersed throughout the rice mitochondrial genome. The total length of these sequences is equal to approximately 6% (22 kb) of the rice mitochondrial genome and to 19% of the chloroplast genome. The transfer of segments of chloroplast DNA seems to have occurred at different times, both before and after the divergence of rice and maize. The mitochondrial genome appears to have been rearranged after the transfer of chloroplast sequences as a result of recombination at these sequences. The rice mitochondrial DNA contains nine intact tRNA genes and three tRNA pseudogenes derived from the chloroplast genome.     

Small, repetitive DNAs contribute significantly to the expanded mitochondrial genome of cucumber

Closely related cucurbit species possess eightfold differences in the sizes of their mitochondrial genomes. We cloned mitochondrial DNA (mtDNA) fragments showing strong hybridization signals to cucumber mtDNA and little or no signal to watermelon mtDNA. The cucumber mtDNA clones carried short (30-53 bp), repetitive DNA motifs that were often degenerate, overlapping, and showed no homology to any sequences currently in the databases. On the basis of dot-blot hybridizations, seven repetitive DNA motifs accounted for >13% (194 kb) of the cucumber mitochondrial genome, equaling >50% of the size of the Arabidopsis mitochondrial genome. Sequence analysis of 136 kb of cucumber mtDNA revealed only 11.2% with significant homology to previously characterized mitochondrial sequences, 2.4% to chloroplast DNA, and 15% to the seven repetitive DNA motifs. The remaining 71.4% of the sequence was unique to the cucumber mitochondrial genome. There was <4% sequence colinearity surrounding the watermelon and cucumber atp9 coding regions, and the much smaller watermelon mitochondrial genome possessed no significant amounts of cucumber repetitive DNAs. Our results demonstrate that the expanded cucumber mitochondrial genome is in part due to extensive duplication of short repetitive sequences, possibly by recombination and/or replication slippage.     

Homology between the ribosomal DNA of Escherichia coli and mitochondrial DNA preparations of maize is principally to sequences other than mitochondrial rRNA genes

E. coli ribosomal DNA has been used to probe maize mitochondrial DNA. It hybridizes primarily with chloroplast ribosomal DNA sequences and with fungal and bacterial sequences which may contaminate the mtDNA preparations. It also hybridizes to the chloroplast 16S ribosomal RNA gene sequence present in the mitochondrial genome (1) as well as to the mitochondrial 18S ribosomal RNA gene sequence. Weak sequence homology was detected between E. coli rDNA and the mitochondrial 26S ribosomal RNA gene.     

Discordant mtDNA and cpDNA phylogenies indicate geographic speciation and reticulation as driving factors for the diversification of the genus Picea

The phylogeny of the genus Picea was investigated by sequencing three loci from the paternally inherited chloroplast genome (trnK, rbcL and trnTLF) and the intron 2 of the maternally transmitted mitochondrial gene nad1 for 35 species. Significant topological differences were found between the trnK tree and the rbcL and trnTLF phylogenetic trees, and between cpDNA and mtDNA phylogenies. None of the phylogenies matched morphological classifications. The mtDNA phylogeny was geographically more structured than cpDNA phylogenies, reflecting the different inheritance of the two cytoplasmic genomes in the Pinaceae and their differential dispersion by seed only and seed and pollen, respectively. Most North American taxa formed a monophyletic group on the mtDNA tree, with topological patterns suggesting geographic speciation by range fragmentation or by dispersal and isolation. Similar patterns were also found among Asian taxa. Such a trend towards geographic speciation is anticipated in other Pinaceae genera with similar life history, autecology and reproductive system. Incongruences between organelle phylogenies suggested the occurrence of mtDNA capture by invading cpDNA. Incongruences between cpDNA partitions further suggested heterologous recombination presumably also linked to ancient reticulate evolution. Whilst cpDNA appears potentially valuable for molecular taxonomy and systematics purposes, these results emphasize the reduced value of cpDNA to infer vertical descent and the speciation history for plants with paternal transmission and high dispersal of their chloroplast genome.     

Extensive Rearrangements in the Chloroplast Genome of <Emphasis Type="Italic">Trachelium caeruleum</Emphasis> Are Associated with Repeats and tRNA Genes

Chloroplast genome organization, gene order, and content are highly conserved among land plants. We sequenced the chloroplast genome of Trachelium caeruleum L. (Campanulaceae), a member of an angiosperm family known for highly rearranged genomes. The total genome size is 162,321 bp, with an inverted repeat (IR) of 27,273 bp, large single-copy (LSC) region of 100,114 bp, and small single-copy (SSC) region of 7,661 bp. The genome encodes 112 different genes, with 17 duplicated in the IR, a tRNA gene (trnI-cau) duplicated once in the LSC region, and a protein-coding gene (psbJ) with two duplicate copies, for a total of 132 putatively intact genes. ndhK may be a pseudogene with internal stop codons, and clpP, ycf1, and ycf2 are so highly diverged that they also may be pseudogenes. ycf15, rpl23, infA, and accD are truncated and likely nonfunctional. The most conspicuous feature of the Trachelium genome is the presence of 18 internally unrearranged blocks of genes inverted or relocated within the genome relative to the ancestral gene order of angiosperm chloroplast genomes. Recombination between repeats or tRNA genes has been suggested as a mechanism of chloroplast genome rearrangements. The Trachelium chloroplast genome shares with Pelargonium and Jasminum both a higher number of repeats and larger repeated sequences in comparison to eight other angiosperm chloroplast genomes, and these are concentrated near rearrangement endpoints. Genes for tRNAs occur at many but not all inversion endpoints, so some combination of repeats and tRNA genes may have mediated these rearrangements.     

The Complete Sequence of the Mitochondrial Genome of Butomus umbellatus – A Member of an Early Branching Lineage of Monocotyledons

In order to study the evolution of mitochondrial genomes in the early branching lineages of the monocotyledons, i.e., the Acorales and Alismatales, we are sequencing complete genomes from a suite of key taxa. As a starting point the present paper describes the mitochondrial genome of Butomus umbellatus (Butomaceae) based on next-generation sequencing data. The genome was assembled into a circular molecule, 450,826 bp in length. Coding sequences cover only 8.2% of the genome and include 28 protein coding genes, four rRNA genes, and 12 tRNA genes. Some of the tRNA genes and a 16S rRNA gene are transferred from the plastid genome. However, the total amount of recognized plastid sequences in the mitochondrial genome is only 1.5% and the amount of DNA transferred from the nucleus is also low. RNA editing is abundant and a total of 557 edited sites are predicted in the protein coding genes. Compared to the 40 angiosperm mitochondrial genomes sequenced to date, the GC content of the Butomus genome is uniquely high (49.1%). The overall similarity between the mitochondrial genomes of Butomus and Spirodela (Araceae), the closest relative yet sequenced, is low (less than 20%), and the two genomes differ in size by a factor 2. Gene order is also largely unconserved. However, based on its phylogenetic position within the core alismatids Butomus will serve as a good reference point for subsequent studies in the early branching lineages of the monocotyledons.     

DISCORDANCE BETWEEN NUCLEAR AND CHLOROPLAST PHYLOGENIES IN THE HEUCHERA GROUP (SAXIFRAGACEAE)

Various factors, including taxon density, sampling error, convergence, and heterogeneity of evolutionary rates, can potentially lead to incongruence between phylogenetic trees based on different genomes. Particularly at the generic level and below, chloroplast capture resulting from hybridization may distort organismal relationships in phylogenetic analyses based on the chloroplast genome, or genes included therein. However, the extent of such discord between chloroplast DNA (cpDNA) trees and those trees based on nuclear genes has rarely been assessed. We therefore used sequences of the internal transcribed spacer regions (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA) to reconstruct phylogenetic relationships among members of the Heuchera group of genera (Saxifragaceae). The Heuchera group presents an important model for the analysis of chloroplast capture and its impact on phylogenetic reconstruction because hybridization is well documented within genera (e.g., Heuchera), and intergeneric hybrids involving six of the nine genera have been reported. An earlier study provided a well-resolved phylogenetic hypothesis for the Heuchera group based on cpDNA restriction-site variation. However, trees based on ITS sequences are discordant with the cpDNA-based tree. Evidence from both morphology and nuclear-encoded allozymes is consistent with the ITS trees, rather than the cpDNA tree, and several points of phylogenetic discord can clearly be attributed to chloroplast capture. Comparison of the organellar and ITS trees also raises the strong likelihood that ancient events of chloroplast capture occurred between lineages during the early diversification of the Heuchera group. Thus, despite the many advantages and widespread use of cpDNA data in phylogeny reconstruction, comparison of relationships based on cpDNA and ITS sequences for the Heuchera group underscores the need for caution in the use of organellar variation for retrieving phylogeny at lower taxonomic levels, particularly in groups noted for hybridization.     

Horizontal gene transfer from a flowering plant to the insular pine Pinus canariensis (Chr. Sm. Ex DC in Buch)

Horizontal gene transfer (HGT) is viewed as very common in the plant mitochondrial (mt) genome, but, to date, only one case of HGT has been found in gymnosperms. Here we report a new case of HGT, in which a mt nad5-1 fragment was transferred from an angiosperm to Pinus canariensis. Quantitative assay and sequence analyses showed that the foreign nad5-1 is located in the mt genome of P. canariensis and is nonfunctional. An extensive survey in the genus Pinus revealed that the angiosperm-derived nad5-1 is restricted to P. canariensis and present across the species'' range. Molecular dating based on chloroplast DNA suggested that the HGT event occurred in the late Miocene after P. canariensis split from its closest relatives, and that the foreign copy became fixed in P. canariensis owing to drift during its colonization of the Canary Islands. The mechanism of this HGT is unclear but it was probably achieved through either direct cell–cell contact or external vectors. Our discovery provides evidence for an important role of HGT in plant mt genome evolution.     

Phylogenetic analyses of two mitochondrial metabolic genes sampled in parallel from angiosperms find fundamental interlocus incongruence

Plant molecular phylogeneticists have supported an analytical approach of combining loci from different genomes, but the combination of mitochondrial sequences with chloroplast and nuclear sequences is potentially problematic. Low substitution rates in mitochondrial genes should decrease saturation, which is especially useful for the study of deep divergences. However, individual mitochondrial loci are insufficiently informative, so that combining congruent loci is necessary. For this study atp1 and cox1 were selected, which are of similar lengths, encode components of the respiratory pathway, and generally lack introns. Thus, these genes might be expected to have similar functional constraints, selection pressures, and evolutionary histories. Strictly parallel sampling of 52 species was achieved as well as six additional composite terminals with representatives from the major angiosperm clades. However, analyses of the separate loci produced strongly incongruent topologies. The source of the incongruence was investigated by validating sequences with questionable affinities, excluding RNA-edited nucleotides, deleting taxa with unexpected phylogenetic associations, and comparing different phylogenetic methods. However, even after potential artifacts were addressed and sites and taxa putatively associated with conflict were excluded, the resulting gene trees for the two mitochondrial loci were still substantially incongruent by all measures examined. Therefore, combining these loci in phylogenetic analysis may be counterproductive to the goal of fully resolving the angiosperm phylogeny.     

An exceptional horizontal gene transfer in plastids: gene replacement by a distant bacterial paralog and evidence that haptophyte and cryptophyte plastids are sisters

Background  

Horizontal gene transfer (HGT) to the plant mitochondrial genome has recently been shown to occur at a surprisingly high rate; however, little evidence has been found for HGT to the plastid genome, despite extensive sequencing. In this study, we analyzed all genes from sequenced plastid genomes to unearth any neglected cases of HGT and to obtain a measure of the overall extent of HGT to the plastid.     

A tRNA Val(GAC) gene of chloroplast origin in sunflower mitochondria is not transcribed

A tRNA^Val (GAC) gene is located in opposite orientation 552 nucleotides (nt) down-stream of the cytochrome oxidase subunit III (coxIII) gene in sunflower mitochondria. The comparison with the homologous chloroplast DNA revealed that the tRNA^Val gene is part of a 417 nucleotides DNA insertion of chloroplast origin in the mitochondrial genome. No tRNA^Val is encoded in monocot mitochondrial DNA (mtDNA), whereas two tRNA^Val species are coded for by potato mtDNA. The mitochondrial genomes of different plant species thus seem to encode unique sets of tRNAs and must thus be competent in importing the missing differing sets of tRNAs.