Genome copy number predicts extreme evolutionary rate variation in plant mitochondrial DNA.

Nuclear and organellar genomes can evolve at vastly different rates despite occupying the same cell. In most bilaterian animals, mitochondrial DNA (mtDNA) evolves faster than nuclear DNA, whereas this trend is generally reversed in plants. However, in some exceptional angiosperm clades, mtDNA substitution rates have increased up to 5,000-fold compared with closely related lineages. The mechanisms responsible for this acceleration are generally unknown. Because plants rely on homologous recombination to repair mtDNA damage, we hypothesized that mtDNA copy numbers may predict evolutionary rates, as lower copy numbers may provide fewer templates for such repair mechanisms. In support of this hypothesis, we found that copy number explains 47% of the variation in synonymous substitution rates of mtDNA across 60 diverse seed plant species representing ~300 million years of evolution. Copy number was also negatively correlated with mitogenome size, which may be a cause or consequence of mutation rate variation. Both relationships were unique to mtDNA and not observed in plastid DNA. These results suggest that homologous recombinational repair plays a role in driving mtDNA substitution rates in plants and may explain variation in mtDNA evolution more broadly across eukaryotes. Our findings also contribute to broader questions about the relationships between mutation rates, genome size, selection efficiency, and the drift-barrier hypothesis.

Divergent evolution of extreme production of variant plant monounsaturated fatty acids.

Metabolic extremes provide opportunities to understand enzymatic and metabolic plasticity and biotechnological tools for novel biomaterial production. We discovered that seed oils of many Thunbergia species contain up to 92% of the unusual monounsaturated petroselinic acid (18:1Δ6), one of the highest reported levels for a single fatty acid in plants. Supporting the biosynthetic origin of petroselinic acid, we identified a Δ6-stearoyl-acyl carrier protein (18:0-ACP) desaturase from Thunbergia laurifolia, closely related to a previously identified Δ6-palmitoyl-ACP desaturase that produces sapienic acid (16:1Δ6)-rich oils in Thunbergia alata seeds. Guided by a T. laurifolia desaturase crystal structure obtained in this study, enzyme mutagenesis identified key amino acids for functional divergence of Δ6 desaturases from the archetypal Δ9-18:0-ACP desaturase and mutations that result in nonnative enzyme regiospecificity. Furthermore, we demonstrate the utility of the T. laurifolia desaturase for the production of unusual monounsaturated fatty acids in engineered plant and bacterial hosts. Through stepwise metabolic engineering, we provide evidence that divergent evolution of extreme petroselinic acid and sapienic acid production arises from biosynthetic and metabolic functional specialization and enhanced expression of specific enzymes to accommodate metabolism of atypical substrates.

Functional Prokaryotic-Like Deoxycytidine Triphosphate Deaminases and Thymidylate Synthase in Eukaryotic Social Amoebae: Vertical, Endosymbiotic, or Horizontal Gene Transfer?

The de novo synthesis of deoxythymidine triphosphate uses several pathways: gram-negative bacteria use deoxycytidine triphosphate deaminase to convert deoxycytidine triphosphate into deoxyuridine triphosphate, whereas eukaryotes and gram-positive bacteria instead use deoxycytidine monophosphate deaminase to transform deoxycytidine monophosphate to deoxyuridine monophosphate. It is then unusual that in addition to deoxycytidine monophosphate deaminases, the eukaryote Dictyostelium discoideum has 2 deoxycytidine triphosphate deaminases (Dcd1Dicty and Dcd2Dicty). Expression of either DcdDicty can fully rescue the slow growth of an Escherichia coli dcd knockout. Both DcdDicty mitigate the hydroxyurea sensitivity of a Schizosaccharomyces pombe deoxycytidine monophosphate deaminase knockout. Phylogenies show that Dcd1Dicty homologs may have entered the common ancestor of the eukaryotic groups of Amoebozoa, Obazoa, Metamonada, and Discoba through an ancient horizontal gene transfer from a prokaryote or an ancient endosymbiotic gene transfer from a mitochondrion, followed by horizontal gene transfer from Amoebozoa to several other unrelated groups of eukaryotes. In contrast, the Dcd2Dicty homologs were a separate horizontal gene transfer from a prokaryote or a virus into either Amoebozoa or Rhizaria, followed by a horizontal gene transfer between them. ThyXDicty, the D. discoideum thymidylate synthase, another enzyme of the deoxythymidine triphosphate biosynthesis pathway, was suggested previously to be acquired from the ancestral mitochondria or by horizontal gene transfer from alpha-proteobacteria. ThyXDicty can fully rescue the E. coli thymidylate synthase knockout, and we establish that it was obtained by the common ancestor of social amoebae not from mitochondria but from a bacterium. We propose horizontal gene transfer and endosymbiotic gene transfer contributed to the enzyme diversity of the deoxythymidine triphosphate synthesis pathway in most social amoebae, many Amoebozoa, and other eukaryotes.

Oligogalactolipid production during cold challenge is conserved in early diverging lineages.

Severe cold, defined as a damaging cold beyond acclimation temperatures, has unique responses, but the signaling and evolution of these responses are not well understood. Production of oligogalactolipids, which is triggered by cytosolic acidification in Arabidopsis (Arabidopsis thaliana), contributes to survival in severe cold. Here, we investigated oligogalactolipid production in species from bryophytes to angiosperms. Production of oligogalactolipids differed within each clade, suggesting multiple evolutionary origins of severe cold tolerance. We also observed greater oligogalactolipid production in control samples than in temperature-challenged samples of some species. Further examination of representative species revealed a tight association between temperature, damage, and oligogalactolipid production that scaled with the cold tolerance of each species. Based on oligogalactolipid production and transcript changes, multiple angiosperm species share a signal of oligogalactolipid production initially described in Arabidopsis, namely cytosolic acidification. Together, these data suggest that oligogalactolipid production is a severe cold response that originated from an ancestral damage response that remains in many land plant lineages and that cytosolic acidification may be a common signaling mechanism for its activation.

Fragaria mitogenomes evolve rapidly in structure but slowly in sequence and incur frequent multinucleotide mutations mediated by microinversions.

Plant mitochondrial DNA has been described as evolving rapidly in structure but slowly in sequence. However, many of the noncoding portions of plant mitogenomes are not homologous among species, raising questions about the rate and spectrum of mutations in noncoding regions. Recent studies have suggested that the lack of homology in noncoding regions could be due to increased sequence divergence. We compared 30 kb of coding and 200 kb of noncoding DNA from 13 sequenced Fragaria mitogenomes, followed by analysis of the rate of sequence divergence, microinversion events and structural variations. Substitution rates in synonymous sites and nongenic sites are nearly identical, suggesting that the genome-wide point mutation rate is generally consistent. A surprisingly high number of large multinucleotide substitutions were detected in Fragaria mitogenomes, which may have resulted from microinversion events and could affect phylogenetic signal and local rate estimates. Fragaria mitogenomes preferentially accumulate deletions relative to insertions and substantial genomic arrangements, whereas mutation rates could positively associate with these sequence and structural changes among species. Together, these observations suggest that plant mitogenomes exhibit low point mutations genome-wide but exceptionally high structural variations, and our results favour a gain-and-loss model for the rapid loss of homology among plant mitogenomes.

Plastid and mitochondrial phylogenomics reveal correlated substitution rate variation in Koenigia (Polygonoideae, Polygonaceae) and a reduced plastome for Koenigia delicatula including loss of all ndh genes.

Koenigia, a genus proposed by Linnaeus, has a contentious taxonomic history. In particular, relationships among species and the circumscription of the genus relative to Aconogonon remain uncertain. To explore phylogenetic relationships of Koenigia with other members of tribe Persicarieae and to establish the timing of major evolutionary diversification events, genome skimming of organellar sequences was used to assemble plastomes and mitochondrial genes from 15 individuals representing 13 species. Most Persicarieae plastomes exhibit a conserved structure and content relative to other flowering plants. However, Koenigia delicatula has lost functional copies of all ndh genes and the intron from atpF. In addition, the rpl32 gene was relocated in the K. delicatula plastome, which likely occurred via overlapping inversions or differential expansion and contraction of the inverted repeat. The highly supported but conflicting relationships between plastome and mitochondrial trees and among gene trees complicates the circumscription of Koenigia, which could be caused by rapid diversification within a short period. Moreover, the plastome and mitochondrial trees revealed correlated variation in substitution rates among Persicarieae species, suggesting a shared underlying mechanism promoting evolutionary rate variation in both organellar genomes. The divergence of dwarf K. delicatula from other Koenigia species may be associated with the well-known Eocene Thermal Maximum 2 or Early Eocene Climatic Optimum event, while diversification of the core-Koenigia clade associates with the Mid-Miocene Climatic Optimum and the uplift of Qinghai-Tibetan Plateau and adjacent areas.

Extensive Shifts from Cis- to Trans-splicing of Gymnosperm Mitochondrial Introns.

Hundreds of plant mitogenomes have been sequenced from angiosperms, but relatively few mitogenomes are available from its sister lineage, gymnosperms. To examine mitogenomic diversity among extant gymnosperms, we generated draft mitogenomes from 11 diverse species and compared them with four previously published mitogenomes. Examined mitogenomes from Pinaceae and cycads retained all 41 protein genes and 26 introns present in the common ancestor of seed plants, whereas gnetophyte and cupressophyte mitogenomes experienced extensive gene and intron loss. In Pinaceae and cupressophyte mitogenomes, an unprecedented number of exons are distantly dispersed, requiring trans-splicing of 50-70% of mitochondrial introns to generate mature transcripts. RNAseq data confirm trans-splicing of these dispersed exons in Pinus. The prevalence of trans-splicing in vascular plant lineages with recombinogenic mitogenomes suggests that genomic rearrangement is the primary cause of shifts from cis- to trans-splicing in plant mitochondria.

The OXA2a Insertase of Arabidopsis Is Required for Cytochrome c Maturation.

In yeast (Saccharomyces cerevisiae) and human (Homo sapiens) mitochondria, Oxidase assembly protein1 (Oxa1) is the general insertase for protein insertion from the matrix side into the inner membrane while Cytochrome c oxidase assembly protein18 (Cox18/Oxa2) is specifically involved in the topogenesis of the complex IV subunit, Cox2. Arabidopsis (Arabidopsis thaliana) mitochondria contain four OXA homologs: OXA1a, OXA1b, OXA2a, and OXA2b. OXA2a and OXA2b are unique members of the Oxa1 superfamily, in that they possess a tetratricopeptide repeat (TPR) domain at their C termini. Here, we determined the role of OXA2a by studying viable mutant plants generated by partial complementation of homozygous lethal OXA2a transfer-DNA insertional mutants using the developmentally regulated ABSCISIC ACID INSENSITIVE3 (ABI3) promoter. The ABI3p:OXA2a plants displayed growth retardation due to a reduction in the steady-state abundances of both c-type cytochromes, cytochrome c 1 and cytochrome c The observed reduction in the steady-state abundance of complex III could be attributed to cytochrome c 1 being one of its subunits. Expression of a soluble heme lyase from an organism with cytochrome c maturation system III could functionally complement the lack of OXA2a. This implies that OXA2a is required for the system I cytochrome c maturation of Arabidopsis. Due to the interaction of OXA2a with Cytochrome c maturation protein CcmF C-terminal-like protein (CCMFC) in a yeast split-ubiquitin based interaction assay, we propose that OXA2a aids in the membrane insertion of CCMFC, which is presumed to form the heme lyase component of the cytochrome c maturation pathway. In contrast with the crucial role played by the TPR domain of OXA2b, the TPR domain of OXA2a is not essential for its functionality.

Plastomes from tribe Plantagineae (Plantaginaceae) reveal infrageneric structural synapormorphies and localized hypermutation for Plantago and functional loss of ndh genes from Littorella.

Tribe Plantagineae (Plantaginaceae) comprises ~ 270 species in three currently recognized genera (Aragoa, Littorella, Plantago), of which Plantago is most speciose. Plantago plastomes exhibit several atypical features including large inversions, expansions of the inverted repeat, increased repetitiveness, intron losses, and gene-specific increases in substitution rate, but the prevalence of these plastid features among species and subgenera is unknown. To assess phylogenetic relationships and plastomic evolutionary dynamics among Plantagineae genera and Plantago subgenera, we generated 25 complete plastome sequences and compared them with existing plastome sequences from Plantaginaceae. Using whole plastome and partitioned alignments, our phylogenomic analyses provided strong support for relationships among major Plantagineae lineages. General plastid features-including size, GC content, intron content, and indels-provided additional support that reinforced major Plantagineae subdivisions. Plastomes from Plantago subgenera Plantago and Coronopus have synapomorphic expansions and inversions affecting the size and gene order of the inverted repeats, and particular genes near the inversion breakpoints exhibit accelerated nucleotide substitution rates, suggesting localized hypermutation associated with rearrangements. The Littorella plastome lacks functional copies of ndh genes, which may be related to an amphibious lifestyle and partial reliance on CAM photosynthesis.

Episodic and guanine-cytosine-biased bursts of intragenomic and interspecific synonymous divergence in Ajugoideae (Lamiaceae) mitogenomes.

Synonymous substitution rates in plant mitochondrial genomes vary by orders of magnitude among species, whereas synonymous rates among genes within a genome are generally consistent. Exceptionally, genes within the Ajuga reptans (Lamiaceae) mitochondrial genome exhibit unprecedented intragenomic heterogeneity in synonymous sequence divergence, but the biological mechanisms underlying this rate variation remain unclear. We tracked the origin and evolutionary trajectory of mitochondrial rate variations by dense sampling in Ajugoideae and found differences in the timing and magnitude of rate acceleration for particular genes. The most divergent genes accelerated earlier, retained a high rate across Ajugoideae, and are generally devoid of RNA editing, whereas moderately diverged genes accelerated later and retained relatively higher RNA editing frequency. The acceleration of mutation rates correlates with increased guanine-cytosine (GC) content, suggesting a key role for GC-biased gene conversion and/or repair after the breakage of ancestral gene clusters.

Zygnema circumcarinatum UTEX 1559 chloroplast and mitochondrial genomes provide insight into land plant evolution.

The complete chloroplast and mitochondrial genomes of Charophyta have shed new light on land plant terrestrialization. Here, we report the organellar genomes of the Zygnema circumcarinatum strain UTEX 1559, and a comparative genomics investigation of 33 plastomes and 18 mitogenomes of Chlorophyta, Charophyta (including UTEX 1559 and its conspecific relative SAG 698-1a), and Embryophyta. Gene presence/absence was determined across these plastomes and mitogenomes. A comparison between the plastomes of UTEX 1559 (157 548 bp) and SAG 698-1a (165 372 bp) revealed very similar gene contents, but substantial genome rearrangements. Surprisingly, the two plastomes share only 85.69% nucleotide sequence identity. The UTEX 1559 mitogenome size is 215 954 bp, the largest among all sequenced Charophyta. Interestingly, this large mitogenome contains a 50 kb region without homology to any other organellar genomes, which is flanked by two 86 bp direct repeats and contains 15 ORFs. These ORFs have significant homology to proteins from bacteria and plants with functions such as primase, RNA polymerase, and DNA polymerase. We conclude that (i) the previously published SAG 698-1a plastome is probably from a different Zygnema species, and (ii) the 50 kb region in the UTEX 1559 mitogenome might be recently acquired as a mobile element.

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.

Lycophyte plastid genomics: extreme variation in GC, gene and intron content and multiple inversions between a direct and inverted orientation of the rRNA repeat.

Lycophytes are a key group for understanding vascular plant evolution. Lycophyte plastomes are highly distinct, indicating a dynamic evolutionary history, but detailed evaluation is hindered by the limited availability of sequences. Eight diverse plastomes were sequenced to assess variation in structure and functional content across lycophytes. Lycopodiaceae plastomes have remained largely unchanged compared with the common ancestor of land plants, whereas plastome evolution in Isoetes and especially Selaginella is highly dynamic. Selaginella plastomes have the highest GC content and fewest genes and introns of any photosynthetic land plant. Uniquely, the canonical inverted repeat was converted into a direct repeat (DR) via large-scale inversion in some Selaginella species. Ancestral reconstruction identified additional putative transitions between an inverted and DR orientation in Selaginella and Isoetes plastomes. A DR orientation does not disrupt the activity of copy-dependent repair to suppress substitution rates within repeats. Lycophyte plastomes include the most archaic examples among vascular plants and the most reconfigured among land plants. These evolutionary trends correlate with the mitochondrial genome, suggesting shared underlying mechanisms. Copy-dependent repair for DR-localized genes indicates that recombination and gene conversion are not inhibited by the DR orientation. Gene relocation in lycophyte plastomes occurs via overlapping inversions rather than transposase/recombinase-mediated processes.

The Reverse Transcriptase/RNA Maturase Protein MatR Is Required for the Splicing of Various Group II Introns in Brassicaceae Mitochondria.

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.

Phylogenomic evidence for ancient recombination between plastid genomes of the Cupressus-Juniperus-Xanthocyparis complex (Cupressaceae).

BACKGROUND: Phylogenetic relationships among Eastern Hemisphere cypresses, Western Hemisphere cypresses, junipers, and their closest relatives are controversial, and generic delimitations have been in flux for the past decade. To address relationships and attempt to produce a more robust classification, we sequenced 11 new plastid genomes (plastomes) from the five variously described genera in this complex (Callitropsis, Cupressus, Hesperocyparis, Juniperus, and Xanthocyparis) and compared them with additional plastomes from diverse members of Cupressaceae. RESULTS: Phylogenetic analysis of protein-coding genes recovered a topology in which Juniperus is sister to Cupressus, whereas a tree based on whole plastomes indicated that the Callitropsis-Hesperocyparis-Xanthocyparis (CaHX) clade is sister to Cupressus. A sliding window analysis of site-specific phylogenetic support identified a ~ 15 kb region, spanning the genes ycf1 and ycf2, which harbored an anomalous signal relative to the rest of the genome. After excluding these genes, trees based on the remainder of the genes and genome consistently recovered a topology grouping the CaHX clade and Cupressus with strong bootstrap support. In contrast, trees based on the ycf1 and ycf2 region strongly supported a sister relationship between Cupressus and Juniperus. CONCLUSIONS: These results demonstrate that standard phylogenomic analyses can result in strongly supported but conflicting trees. We suggest that the conflicting plastomic signals result from an ancient introgression event involving ycf1 and ycf2 that occurred in an ancestor of this species complex. The introgression event was facilitated by plastomic recombination in an ancestral heteroplasmic individual carrying distinct plastid haplotypes, offering further evidence that recombination occurs between plastomes. Finally, we provide strong support for previous proposals to recognize five genera in this species complex: Callitropsis, Cupressus, Hesperocyparis, Juniperus, and Xanthocyparis.

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.

Complete mitochondrial genomes from the ferns Ophioglossum californicum and Psilotum nudum are highly repetitive with the largest organellar introns.

Currently, complete mitochondrial genomes (mitogenomes) are available from all major land plant lineages except ferns. Sequencing of fern mitogenomes could shed light on the major evolutionary transitions that established mitogenomic diversity among extant lineages. In this study, we generated complete mitogenomes from the adder's tongue fern (Ophioglossum californicum) and the whisk fern (Psilotum nudum). The Psilotum mitogenome (628 kb) contains a rich complement of genes and introns, some of which are the largest of any green plant organellar genome. In the Ophioglossum mitogenome (372 kb), gene and intron content is slightly reduced, including the loss of all four mitochondrial ccm genes. Transcripts of nuclear Ccm genes also were not detected, suggesting loss of the entire mitochondrial cytochrome c maturation pathway from Ophioglossum. Both fern mitogenomes are highly repetitive, yet they show extremely low levels of active recombination. Transcriptomic sequencing uncovered ˜1000 sites of C-to-U RNA editing in both species, plus a small number (< 60) of U-to-C edit sites. Overall, the first mitochondrial genomes of ferns show a mix of features shared with lycophytes and/or seed plants and several novel genomic features, enabling a robust reconstruction of the mitogenome in the common ancestor of vascular plants.

Evolutionary dynamics of the plastid inverted repeat: the effects of expansion, contraction, and loss on substitution rates.

Rates of nucleotide substitution were previously shown to be several times slower in the plastid inverted repeat (IR) compared with single-copy (SC) regions, suggesting that the IR provides enhanced copy-correction activity. To examine the generality of this synonymous rate dependence on the IR, we compared plastomes from 69 pairs of closely related species representing 52 families of angiosperms, gymnosperms, and ferns. We explored the breadth of IR boundary shifts in land plants and demonstrate that synonymous substitution rates are, on average, 3.7 times slower in IR genes than in SC genes. In addition, genes moved from the SC into the IR exhibit lower synonymous rates consistent with other IR genes, while genes moved from the IR into the SC exhibit higher rates consistent with other SC genes. Surprisingly, however, several plastid genes from Pelargonium, Plantago, and Silene have highly accelerated synonymous rates despite their IR localization. Together, these results provide strong evidence that the duplicative nature of the IR reduces the substitution rate within this region. The anomalously fast-evolving genes in Pelargonium, Plantago, and Silene indicate localized hypermutation, potentially induced by a higher level of error-prone double-strand break repair in these regions, which generates substitutional rate variation.

Variable presence of the inverted repeat and plastome stability in Erodium.

BACKGROUND AND AIMS: Several unrelated lineages such as plastids, viruses and plasmids, have converged on quadripartite genomes of similar size with large and small single copy regions and a large inverted repeat (IR). Except for Erodium (Geraniaceae), saguaro cactus and some legumes, the plastomes of all photosynthetic angiosperms display this structure. The functional significance of the IR is not understood and Erodium provides a system to examine the role of the IR in the long-term stability of these genomes. We compared the degree of genomic rearrangement in plastomes of Erodium that differ in the presence and absence of the IR. METHODS: We sequenced 17 new Erodium plastomes. Using 454, Illumina, PacBio and Sanger sequences, 16 genomes were assembled and categorized along with one incomplete and two previously published Erodium plastomes. We conducted phylogenetic analyses among these species using a dataset of 19 protein-coding genes and determined if significantly higher evolutionary rates had caused the long branch seen previously in phylogenetic reconstructions within the genus. Bioinformatic comparisons were also performed to evaluate plastome evolution across the genus. KEY RESULTS: Erodium plastomes fell into four types (Type 1-4) that differ in their substitution rates, short dispersed repeat content and degree of genomic rearrangement, gene and intron content and GC content. Type 4 plastomes had significantly higher rates of synonymous substitutions (dS) for all genes and for 14 of the 19 genes non-synonymous substitutions (dN) were significantly accelerated. We evaluated the evidence for a single IR loss in Erodium and in doing so discovered that Type 4 plastomes contain a novel IR. CONCLUSIONS: The presence or absence of the IR does not affect plastome stability in Erodium. Rather, the overall repeat content shows a negative correlation with genome stability, a pattern in agreement with other angiosperm groups and recent findings on genome stability in bacterial endosymbionts.

Unprecedented heterogeneity in the synonymous substitution rate within a plant genome.

The synonymous substitution rate varies widely among species, but it is generally quite stable within a genome due to the absence of strong selective pressures. In plants, plastid genes tend to evolve faster than mitochondrial genes, rate variation among species generally correlates between the mitochondrial and plastid genomes, and few examples of intragenomic rate heterogeneity exist. To study the extent of substitution rate variation between and within plant organellar genomes, we sequenced the complete mitochondrial and plastid genomes from the bugleweed, Ajuga reptans, which was previously shown to exhibit rate heterogeneity for several mitochondrial genes. Substitution rates were accelerated specifically in the mitochondrial genome, which contrasts with correlated plastid and mitochondrial rate changes in most other angiosperms. Strikingly, we uncovered a 340-fold range of synonymous substitution rate variation among Ajuga mitochondrial genes. This is by far the largest amount of synonymous rate heterogeneity ever reported for a genome, but the evolutionary forces driving this phenomenon are unclear. Selective effects on synonymous sites in plant mitochondria are generally weak and thus unlikely to generate such unprecedented intragenomic rate heterogeneity. Quickly evolving genes are not clustered in the genome, arguing against localized hypermutation, although it is possible that they were clustered ancestrally given the high rate of genomic rearrangement in plant mitochondria. Mutagenic retroprocessing, involving error-prone reverse transcription and genomic integration of mature transcripts, is hypothesized as another potential explanation.