The Complete Mitochondrial Genome Sequence of the Hornwort <Emphasis Type="Italic">Megaceros</Emphasis><Emphasis Type="Italic">aenigmaticus</Emphasis> Shows a Mixed Mode of Conservative Yet Dynamic Evolution in Early Land Plant Mitochondrial Genomes

Land plants possess some of the most unusual mitochondrial genomes among eukaryotes. However, in early land plants these genomes resemble those of green and red algae or early eukaryotes. The question of when during land plant evolution the dramatic change in mtDNAs occurred remains unanswered. Here we report the first completely sequenced mitochondrial genome of the hornwort, Megaceros aenigmaticus, a member of the sister group of vascular plants. It is a circular molecule of 184,908 base pairs, with 32 protein genes, 3 rRNA genes, 17 tRNA genes, and 30 group II introns. The genome contains many genes arranged in the same order as in those of a liverwort, a moss, several green and red algae, and Reclinomonas americana, an early-branching eukaryote with the most ancestral form of mtDNA. In particular, the gene order between mtDNAs of the hornwort and Physcomitrella patens (moss) differs by only 8 inversions and translocations. However, the hornwort mtDNA possesses 4 derived features relative to green alga mtDNAs—increased genome size, RNA editing, intron gains, and gene losses—which were all likely acquired during the origin and early evolution of land plants. Overall, this genome and those of other 2 bryophytes show that mitochondrial genomes in early land plants, unlike their seed plant counterparts, exhibit a mixed mode of conservative yet dynamic evolution. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Libo Li and Bin Wang contributed equally to this work.     

The chloroplast genome from a lycophyte (microphyllophyte), <Emphasis Type="Italic">Selaginella uncinata</Emphasis>, has a unique inversion,transpositions and many gene losses

We determined the complete nucleotide sequence of the chloroplast genome of Selaginella uncinata, a lycophyte belonging to the basal lineage of the vascular plants. The circular double-stranded DNA is 144,170 bp, with an inverted repeat of 25,578 bp separated by a large single copy region (LSC) of 77,706 bp and a small single copy region (SSC) of 40,886 bp. We assigned 81 protein-coding genes including four pseudogenes, four rRNA genes and only 12 tRNA genes. Four genes, rps15, rps16, rpl32 and ycf10, found in most chloroplast genomes in land plants were not present in S. uncinata. While gene order and arrangement of the chloroplast genome of another lycophyte, Hupertzia lucidula, are almost the same as those of bryophytes, those of S. uncinata differ considerably from the typical structure of bryophytes with respect to the presence of a unique 20 kb inversion within the LSC, transposition of two segments from the LSC to the SSC and many gene losses. Thus, the organization of the S. uncinata chloroplast genome provides a new insight into the evolution of lycophytes, which were separated from euphyllophytes approximately 400 million years ago. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mosses share mitochondrial group II introns with flowering plants, not with liverworts

Extant bryophytes are regarded as the closest living relatives of the first land plants, but relationships among the bryophyte classes (mosses, liverworts and hornworts) and between them and other embryophytes have remained unclear. We have recently found that plant mitochondrial genes with positionally stable introns are well suited for addressing questions of plant phylogeny at a deep level. To explore further data sets we have chosen to investigate the mitochondrial genes nad4 and nad7, which are particularly rich in intron sequences. Surprisingly, we find that in these genes mosses share three group II introns with flowering plants, but none with the liverwort Marchantia polymorpha or other liverworts investigated here. In mitochondria of Marchantia, nad7 is a pseudogene containing stop codons, but nad7 appears as a functional mitochondrial gene in mosses, including the isolated genus Takakia. We observe the necessity for strikingly frequent C-to-U RNA editing to reconstitute conserved codons in Takakia when compared to other mosses. The findings underline the great evolutionary distances among the bryophytes as the presumptive oldest division of land plants. A scenario involving differential intron gains from fungal sources in what are perhaps the two earliest diverging land plant lineages, liverworts and other embryophytes, is discussed. With their positionally stable introns, nad4 and nad7 represent novel marker genes that may permit a detailed phylogenetic resolution of early clades of land plants.     

Discovery of the rpl10 Gene in Diverse Plant Mitochondrial Genomes and Its Probable Replacement by the Nuclear Gene for Chloroplast RPL10 in Two Lineages of Angiosperms

Mitochondrial genomes of plants are much larger than those of mammals and often contain conserved open reading frames (ORFs) of unknown function. Here, we show that one of these conserved ORFs is actually the gene for ribosomal protein L10 (rpl10) in plant. No rpl10 gene has heretofore been reported in any mitochondrial genome other than the exceptionally gene-rich genome of the protist Reclinomonas americana. Conserved ORFs corresponding to rpl10 are present in a wide diversity of land plant and green algal mitochondrial genomes. The mitochondrial rpl10 genes are transcribed in all nine land plants examined, with five seed plant genes subject to RNA editing. In addition, mitochondrial-rpl10-like cDNAs were identified in EST libraries from numerous land plants. In three lineages of angiosperms, rpl10 is either lost from the mitochondrial genome or a pseudogene. In two of them (Brassicaceae and monocots), no nuclear copy of mitochondrial rpl10 is identifiably present, and instead a second copy of nuclear-encoded chloroplast rpl10 is present. Transient assays using green fluorescent protein indicate that this duplicate gene is dual targeted to mitochondria and chloroplasts. We infer that mitochondrial rpl10 has been functionally replaced by duplicated chloroplast counterparts in Brassicaceae and monocots.     

Complete Chloroplast Genome of Sedum sarmentosum and Chloroplast Genome Evolution in Saxifragales

Comparative chloroplast genome analyses are mostly carried out at lower taxonomic levels, such as the family and genus levels. At higher taxonomic levels, chloroplast genomes are generally used to reconstruct phylogenies. However, little attention has been paid to chloroplast genome evolution within orders. Here, we present the chloroplast genome of Sedum sarmentosum and take advantage of several available (or elucidated) chloroplast genomes to examine the evolution of chloroplast genomes in Saxifragales. The chloroplast genome of S. sarmentosum is 150,448 bp long and includes 82,212 bp of a large single-copy (LSC) region, 16.670 bp of a small single-copy (SSC) region, and a pair of 25,783 bp sequences of inverted repeats (IRs).The genome contains 131 unique genes, 18 of which are duplicated within the IRs. Based on a comparative analysis of chloroplast genomes from four representative Saxifragales families, we observed two gene losses and two pseudogenes in Paeonia obovata, and the loss of an intron was detected in the rps16 gene of Penthorum chinense. Comparisons among the 72 common protein-coding genes confirmed that the chloroplast genomes of S. sarmentosum and Paeonia obovata exhibit accelerated sequence evolution. Furthermore, a strong correlation was observed between the rates of genome evolution and genome size. The detected genome size variations are predominantly caused by the length of intergenic spacers, rather than losses of genes and introns, gene pseudogenization or IR expansion or contraction. The genome sizes of these species are negatively correlated with nucleotide substitution rates. Species with shorter duration of the life cycle tend to exhibit shorter chloroplast genomes than those with longer life cycles.     

Evidence for the most basal split in land plants dividing bryophyte and tracheophyte lineages

The problem of relationships among the major basal living groups of land plants is long standing, yet the uncertainty as to the phylogenetic affinity of these lines persists in the literature. Molecular and modern cladistic studies of the phylogenetic relationships of the above groups resulted in a large number of conflicting topologies. However, with the exception of the cladistic analyses of spermatogenesis, suggesting monophyly of extant bryophytes, these studies agree the paraphyletic bryophyte grade is basal within the embryophyte tree. Here we would like to present analyses on the basis of the concatenated datasets of nucleotide and amino-acid sequences of 57 protein-coding genes common to 17 chloroplast genomes of land plants and a charophyte alga Chaetosphaeridium globosum. Character-wise, these are the largest datasets currently available to address the problem of basal relationships within embryophytes. Main lineages of bryophytes, i.e liverworts, hornworts and mosses are represented in our alignments with a single taxon, whereas 14 taxa represent the tracheophytes. With our data, phylogeny with liverwort basal appears to be and artifact related to high and unequal A+T contents among the sequences analysed. Reducing this compositional bias and applying methods developed to counter it, we recovered an alternative, strongly supported topology wherein both bryophytes and tracheophytes are monophyletic. Within bryophytes, hornworts are basal and liverworts are sister to mosses.     

Tobacco plastid ribosomal protein S18 is essential for cell survival

Plastid genomes contain a conserved set of genes most of which are involved in either photosynthesis or gene expression. Among the ribosomal protein genes present in higher plant plastid genomes, rps18 is special in that it is absent from the plastid genomes of several non-green unicellular organisms, including Euglena longa and Toxoplasma gondii. Here we have tested whether the ribosomal protein S18 is required for translation by deleting the rps18 gene from the tobacco plastid genome. We report that, while deletion of the rps18 gene was readily obtained, no homoplasmic Δrps18 plants or leaf sectors could be isolated. Instead, segregation into homoplasmy led to severe defects in leaf development suggesting that the knockout of rps18 is lethal and the S18 protein is required for cell survival. Our data demonstrate that S18 is indispensable for plastid ribosome function in tobacco and support an essential role for plastid translation in plant development. Moreover, we demonstrate the occurrence of flip-flop recombination on short inverted repeat sequences which generates different isoforms of the transformed plastid genome that differ in the orientation a 70 kb segment in the large single-copy region. However, infrequent occurrence of flip-flop recombination and random segregation of plastid genomes result in the predominant presence of only one of the isoforms in many tissue samples. Implications for the interpretation of chloroplast transformation experiments and vector design are discussed.     

Chloroplast phylogeny indicates that bryophytes are monophyletic

Opinions on the basal relationship of land plants vary considerably and no phylogenetic tree with significant statistical support has been obtained. Here, we report phylogenetic analyses using 51 genes from the entire chloroplast genome sequences of 20 representative green plant species. The analyses, using translated amino acid sequences, indicated that extant bryophytes (mosses, liverworts, and hornworts) form a monophyletic group with high statistical confidence and that extant bryophytes are likely sisters to extant vascular plants, although the support for monophyletic vascular plants was not strong. Analyses at the nucleotide level could not resolve the basal relationship with statistical confidence. Bryophyte monophyly inferred using amino acid sequences has a good statistical foundation and is not rejected statistically by other data sets. We propose bryophyte monophyly as the currently best hypothesis.     

THE RPS12 GENE IN SPROGYRA MAXIMA (CHLOROPHYTA) AND ITS EVOLUTIONARY SINGNIFICANCE1

The str operon consists fo four genes in eubacteria. Portions of his operon are conserved in the chloroplast genomes of green algae and land plants. In land plant chloroplasts, the str operon comprises only two genes, rps12 and rps7, and is arranged in a trans-spliced state. Since no other previously studied chloroplast genome contains this arrangement, and because the charophyte lineage is the sister group of land plants, we chose to look for this arrangement in the Charophyceae. The two str genes, rps12 and rps7, present in the chloroplasts of Spirogyra maxima Hanssall, were identified by hybridization of a Southern blot and requenced. The results indicate that Spirogyra contains a str operon almost identical to that of land plant chloroplasts. Based upon the structure of the operon in other chloroplasts and eubacterial genomes, the trans-spliced state most likely evolved early within the charophyte lineage.     

Plant Mitochondrial RNA Editing

RNA editing affects messenger RNAs and transfer RNAs in plant mitochondria by site-specific exchange of cytidine and uridine bases in both seed and nonseed plants. Distribution of the phenomenon among bryophytes has been unclear since RNA editing has been detected in some but not all liverworts and mosses. A more detailed understanding of RNA editing in plants required extended data sets for taxa and sequences investigated. Toward this aim an internal region of the mitochondrial nad5 gene (1104 nt) was analyzed in a large collection of bryophytes and green algae (Charales). The genomic nad5 sequences predict editing in 30 mosses, 2 hornworts, and 7 simple thalloid and leafy liverworts (Jungermanniidae). No editing is, however, required in seven species of the complex thalloid liverworts (Marchantiidae) and the algae. RNA editing among the Jungermanniidae, on the other hand, reaches frequencies of up to 6% of codons being modified. Predictability of RNA editing from the genomic sequences was confirmed by cDNA analysis in the mosses Schistostega pennata and Rhodobryum roseum, the hornworts Anthoceros husnotii and A. punctatus, and the liverworts Metzgeria conjugata and Moerckia flotoviana. All C-to-U nucleotide exchanges predicted to reestablish conserved codons were confirmed. Editing in the hornworts includes the removal of genomic stop codons by frequent reverse U-to-C edits. Expectedly, no RNA editing events were identified by cDNA analysis in the marchantiid liverworts Ricciocarpos natans, Corsinia coriandra, and Lunularia cruciata. The findings are discussed in relation to models on the phylogeny of land plants. Received: 2 April 1998 / Accepted: 4 August 1998     

Six newly sequenced chloroplast genomes from prasinophyte green algae provide insights into the relationships among prasinophyte lineages and the diversity of streamlined genome architecture in picoplanktonic species

Background

Because they represent the earliest divergences of the Chlorophyta, the morphologically diverse unicellular green algae making up the prasinophytes hold the key to understanding the nature of the first viridiplants and the evolutionary patterns that accompanied the radiation of chlorophytes. Nuclear-encoded 18S rDNA phylogenies unveiled nine prasinophyte clades (clades I through IX) but their branching order is still uncertain. We present here the newly sequenced chloroplast genomes of Nephroselmis astigmatica (clade III) and of five picoplanktonic species from clade VI (Prasinococcus sp. CCMP 1194, Prasinophyceae sp. MBIC 106222 and Prasinoderma coloniale) and clade VII (Picocystis salinarum and Prasinophyceae sp. CCMP 1205). These chloroplast DNAs (cpDNAs) were compared with those of the six previously sampled prasinophytes (clades I, II, III and V) in order to gain information both on the relationships among prasinophyte lineages and on chloroplast genome evolution.

Results

Varying from 64.3 to 85.6 kb in size and encoding 100 to 115 conserved genes, the cpDNAs of the newly investigated picoplanktonic species are substantially smaller than those observed for larger-size prasinophytes, are economically packed and contain a reduced gene content. Although the Nephroselmis and Picocystis cpDNAs feature a large inverted repeat encoding the rRNA operon, gene partitioning among the single copy regions is remarkably different. Unexpectedly, we found that all three species from clade VI (Prasinococcales) harbor chloroplast genes not previously documented for chlorophytes (ndhJ, rbcR, rpl21, rps15, rps16 and ycf66) and that Picocystis contains a trans-spliced group II intron. The phylogenies inferred from cpDNA-encoded proteins are essentially congruent with 18S rDNA trees, resolving with robust support all six examined prasinophyte lineages, with the exception of the Pycnococcaceae.

Conclusions

Our results underscore the high variability in genome architecture among prasinophyte lineages, highlighting the strong pressure to maintain a small and compact chloroplast genome in picoplanktonic species. The unique set of six chloroplast genes found in the Prasinococcales supports the ancestral status of this lineage within the prasinophytes. The widely diverging traits uncovered for the clade-VII members (Picocystis and Prasinophyceae sp. CCMP 1205) are consistent with their resolution as separate lineages in the chloroplast phylogeny.     

Substitution of the gene for chloroplast RPS16 was assisted by generation of a dual targeting signal

Organelle (mitochondria and chloroplasts in plants) genomes lost a large number of genes after endosymbiosis occurred. Even after this major gene loss, organelle genomes still lose their own genes, even those that are essential, via gene transfer to the nucleus and gene substitution of either different organelle origin or de novo genes. Gene transfer and substitution events are important processes in the evolution of the eukaryotic cell. Gene loss is an ongoing process in the mitochondria and chloroplasts of higher plants. The gene for ribosomal protein S16 (rps16) is encoded in the chloroplast genome of most higher plants but not in Medicago truncatula and Populus alba. Here, we show that these 2 species have compensated for loss of the rps16 from the chloroplast genome by having a mitochondrial rps16 that can target the chloroplasts as well as mitochondria. Furthermore, in Arabidopsis thaliana, Lycopersicon esculentum, and Oryza sativa, whose chloroplast genomes encode the rps16, we show that the product of the mitochondrial rps16 has dual targeting ability. These results suggest that the dual targeting of RPS16 to the mitochondria and chloroplasts emerged before the divergence of monocots and dicots (140-150 MYA). The gene substitution of the chloroplast rps16 by the nuclear-encoded rps16 in higher plants is the first report about ongoing gene substitution by dual targeting and provides evidence for an intermediate stage in the formation of this heterogeneous organelle.     

Complete Chloroplast Genome Sequence of Poisonous and Medicinal Plant Datura stramonium: Organizations and Implications for Genetic Engineering

Datura stramonium is a widely used poisonous plant with great medicinal and economic value. Its chloroplast (cp) genome is 155,871 bp in length with a typical quadripartite structure of the large (LSC, 86,302 bp) and small (SSC, 18,367 bp) single-copy regions, separated by a pair of inverted repeats (IRs, 25,601 bp). The genome contains 113 unique genes, including 80 protein-coding genes, 29 tRNAs and four rRNAs. A total of 11 forward, 9 palindromic and 13 tandem repeats were detected in the D. stramonium cp genome. Most simple sequence repeats (SSR) are AT-rich and are less abundant in coding regions than in non-coding regions. Both SSRs and GC content were unevenly distributed in the entire cp genome. All preferred synonymous codons were found to use A/T ending codons. The difference in GC contents of entire genomes and of the three-codon positions suggests that the D. stramonium cp genome might possess different genomic organization, in part due to different mutational pressures. The five most divergent coding regions and four non-coding regions (trnH-psbA, rps4-trnS, ndhD-ccsA, and ndhI-ndhG) were identified using whole plastome alignment, which can be used to develop molecular markers for phylogenetics and barcoding studies within the Solanaceae. Phylogenetic analysis based on 68 protein-coding genes supported Datura as a sister to Solanum. This study provides valuable information for phylogenetic and cp genetic engineering studies of this poisonous and medicinal plant.     

Primary cell wall composition of bryophytes and charophytes

Major differences in primary cell wall (PCW) components between non-vascular plant taxa are reported. (1) Xyloglucan: driselase digestion yielded isoprimeverose (the diagnostic repeat unit of xyloglucan) from PCW-rich material of Anthoceros (a hornwort), mosses and both leafy and thalloid liverworts, as well as numerous vascular plants, showing xyloglucan to be a PCW component in all land plants tested. In contrast, charophycean green algae (Klebsormidium flaccidium, Coleochaete scutata and Chara corallina), thought to be closely related to land plants, did not contain xyloglucan. They did not yield isoprimeverose; additionally, charophyte material was not digestible with xyloglucan-specific endoglucanase or cellulase to give xyloglucan-derived oligosaccharides. (2) Uronic acids: acid hydrolysis of PCW-rich material from the charophytes, the hornwort, thalloid and leafy liverworts and a basal moss yielded higher concentrations of glucuronic acid than that from the remaining land plants including the less basal mosses and all vascular plants tested. Polysaccharides of the hornwort Anthoceros contained an unusual repeat-unit, glucuronic acid-alpha(1-->3)-galactose, not found in appreciable amounts in any other plants tested. Galacturonic acid was consistently the most abundant PCW uronic acid, but was present in higher concentrations in acid hydrolysates of bryophytes and charophytes than in those of any of the vascular plants. Mannuronic acid was not detected in any of the species surveyed. (3) Mannose: acid hydrolysis of charophyte and bryophyte PCW-rich material also yielded appreciably higher concentrations of mannose than are found in vascular plant PCWs. (4) Mixed-linkage glucan (MLG) was absent from all algae and bryophytes tested; however, upon digestion with licheninase, PCW-rich material from the alga Ulva lactuca and the leafy liverwort Lophocolea bidentata yielded penta- to decasaccharides, indicating the presence of MLG-related polysaccharides. Our results show that major evolutionary events are often associated with changes in PCW composition. In particular, the acquisition of xyloglucan may have been a pre-adaptive advantage that allowed colonization of land.     

Comparative genomics of four Liliales families inferred from the complete chloroplast genome sequence of Veratrum patulum O. Loes. (Melanthiaceae)

The sequence of the chloroplast genome, which is inherited maternally, contains useful information for many scientific fields such as plant systematics, biogeography and biotechnology because its characteristics are highly conserved among species. There is an increase in chloroplast genomes of angiosperms that have been sequenced in recent years. In this study, the nucleotide sequence of the chloroplast genome (cpDNA) of Veratrum patulum Loes. (Melanthiaceae, Liliales) was analyzed completely. The circular double-stranded DNA of 153,699 bp consists of two inverted repeat (IR) regions of 26,360 bp each, a large single copy of 83,372 bp, and a small single copy of 17,607 bp. This plastome contains 81 protein-coding genes, 30 distinct tRNA and four genes of rRNA. In addition, there are six hypothetical coding regions (ycf1, ycf2, ycf3, ycf4, ycf15 and ycf68) and two open reading frames (ORF42 and ORF56), which are also found in the chloroplast genomes of the other species. The gene orders and gene contents of the V. patulum plastid genome are similar to that of Smilax china, Lilium longiflorum and Alstroemeria aurea, members of the Smilacaceae, Liliaceae and Alstroemeriaceae (Liliales), respectively. However, the loss rps16 exon 2 in V. patulum results in the difference in the large single copy regions in comparison with other species. The base substitution rate is quite similar among genes of these species. Additionally, the base substitution rate of inverted repeat region was smaller than that of single copy regions in all observed species of Liliales. The IR regions were expanded to trnH_GUG in V. patulum, a part of rps19 in L. longiflorum and A. aurea, and whole sequence of rps19 in S. china. Furthermore, the IGS lengths of rbcL-accD-psaI region were variable among Liliales species, suggesting that this region might be a hotspot of indel events and the informative site for phylogenetic studies in Liliales. In general, the whole chloroplast genome of V. patulum, a potential medicinal plant, will contribute to research on the genetic applications of this genus.     

The Chloroplast Genome Sequence of Mungbean (Vigna radiata) Determined by High-throughput Pyrosequencing: Structural Organization and Phylogenetic Relationships

Mungbean is an economically important crop which is grown principally for its protein-rich dry seeds. However, genomic research of mungbean has lagged behind other species in the Fabaceae family. Here, we reported the complete chloroplast (cp) genome sequence of mungbean obtained by the 454 pyrosequencing technology. The mungbean cp genome is 151 271 bp in length which includes a pair of inverted repeats (IRs) of 26 474 bp separated by a small single-copy region of 17 427 bp and a large single-copy region of 80 896 bp. The genome contains 108 unique genes and 19 of these genes are duplicated in the IR. Of these, 75 are predicted protein-coding genes, 4 ribosomal RNA genes and 29 tRNA genes. Relative to other plant cp genomes, we observed two distinct rearrangements: a 50-kb inversion between accD/rps16 and rbcL/trnK-UUU, and a 78-kb rearrangement between trnH/rpl14 and rps19/rps8. We detected sequence length polymorphism in the cp homopolymeric regions at the intra- and inter-specific levels in the Vigna species. Phylogenetic analysis demonstrated a close relationship between Vigna and Phaseolus in the phaseolinae subtribe and provided a strong support for a monophyletic group of the eurosid I.     

The complete mitochondrial DNA sequence of Mesostigma viride identifies this green alga as the earliest green plant divergence and predicts a highly compact mitochondrial genome in the ancestor of all green plants.

To gain insights into the nature of the mitochondrial genome in the common ancestor of all green plants, we have completely sequenced the mitochondrial DNA (mtDNA) of Mesostigma viride. This green alga belongs to a morphologically heterogeneous class (Prasinophyceae) that includes descendants of the earliest diverging green plants. Recent phylogenetic analyses of ribosomal RNAs (rRNAs) and concatenated proteins encoded by the chloroplast genome identified Mesostigma as a basal branch relative to the Streptophyta and the Chlorophyta, the two phyla that were previously thought to contain all extant green plants. The circular mitochondrial genome of Mesostigma resembles the mtDNAs of green algae occupying a basal position within the Chlorophyta in displaying a small size (42,424 bp) and a high gene density (86.6% coding sequences). It contains 65 genes that are conserved in other mtDNAs. Although none of these genes represents a novel coding sequence among green plant mtDNAs, four of them (rps1, sdh3, sdh4, and trnL[caa]) have not been reported previously in chlorophyte mtDNAs, and two others (rpl14 and trnI[gau]) have not been identified in the streptophyte mtDNAs examined so far (land-plant mtDNAs). Phylogenetic analyses of 19 concatenated mtDNA-encoded proteins favor the hypothesis that Mesostigma represents the earliest branch of green plant evolution. Four group I introns (two in rnl and two in cox1) and three group II introns (two in nad3 and one in cox2), two of which are trans-spliced at the RNA level, reside in Mesostigma mtDNA. The insertion sites of the three group II introns are unique to this mtDNA, suggesting that trans-splicing arose independently in the Mesostigma lineage and in the Streptophyta. The few structural features that can be regarded as ancestral in Mesostigma mtDNA predict that the common ancestor of all green plants had a compact mtDNA containing a minimum of 75 genes and perhaps two group I introns. Considering that the mitochondrial genome is much larger in size in land plants than in Mesostigma, we infer that mtDNA size began to increase dramatically in the Streptophyta either during the evolution of charophyte green algae or during the transition from charophytes to land plants.