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

Monique Turmel, Christian Otis and Claude Lemieux

Canadian Institute for Advanced Research, Département de Biochimie et de Microbiologie, Université Laval, Québec, Canada


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
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.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
Molecular phylogenies inferred from nuclear small-subunit ribosomal DNA sequences (Melkonian, Marin, and Surek 1995Citation ; Friedl 1997Citation ; Chapman et al. 1998Citation ) suggest that all green plants (Viridiplantae sensu Cavalier-Smith [1981Citation ]) belong to one of two phyla, Streptophyta (land plants and their closest green algal ancestors, the charophytes) and Chlorophyta (the rest of the green algae which comprise members of the Chlorophyceae, the Trebouxiophyceae, the Ulvophyceae, and the nonmonophyletic class Prasinophyceae). But this notion has been challenged by the recent analysis of the complete chloroplast DNA (cpDNA) sequence from the prasinophyte Mesostigma viride (Lemieux, Otis, and Turmel 2000Citation ). In trees inferred from multiple cpDNA-encoded proteins and ribosomal RNAs (rRNAs), Mesostigma represents a strongly supported branch that is basal relative to the Streptophyta and the Chlorophyta, a position independently supported by structural and organizational features of Mesostigma cpDNA. Previously reported trees inferred from actin (Bhattacharya et al. 1998Citation ) and nuclear small-subunit rRNA sequences (Marin and Melkonian 1999Citation ) had placed Mesostigma within the Streptophyta, a position consistent with the identical orientation of multilayered structures relative to flagellar roots in Mesostigma and charophytes (Melkonian 1989Citation ). The apparent conflict between the chloroplast and the nuclear trees is possibly caused by the inability of the nuclear markers analyzed to provide a strong phylogenetic signal. Whereas the actin sequences cannot discriminate between the hypotheses that Mesostigma is positioned within the Chlorophyta or at the base of the Chlorophyta and the Streptophyta (Lemieux, Otis, and Turmel 2000Citation ), the nuclear small-subunit rRNA sequences fail to resolve the divergence order of basal streptophyte lineages and cannot provide strong bootstrap support for the monophyly of the Streptophyta (Marin and Melkonian 1999Citation ). Alternatively, the conflict between chloroplast and nuclear trees can possibly be explained by the small number of green plant taxa (three streptophytes, all of which are land plants, and three chlorophytes) that were used as sources of the chloroplast data. This limited taxon sampling led to the suggestion (Qiu and Lee 2000)Citation that the basal position of Mesostigma was the result of tree-construction artifacts. Given that the branch bearing the Mesostigma sequence was very short, long-branch attraction artifacts (Felsenstein 1988Citation ) do not appear to be the cause of the conflict.

The Mesostigma cpDNA sequence not only provides important insights into the evolutionary relationships among green plants, but also into the nature of the chloroplast genome in the common ancestor of all green plants (Lemieux, Otis, and Turmel 2000Citation ). Mesostigma cpDNA features numerous ancestral characters and at the gene organizational level more highly resembles its land plant homologs than its chlorophyte homologs, suggesting that the ancestral green algal chloroplast genome has undergone rather limited rearrangements during the long evolutionary period leading to land plants. Also, the gene structure and the gene content of Mesostigma cpDNA predict that this ancestral genome did not have any introns and featured five extra genes in addition to those present in the streptophyte and the chlorophyte cpDNAs examined thus far.

To test the hypothesis that Mesostigma represents the earliest branch of green plant evolution and also to gain insights into the nature of the mitochondrial genome in the common ancestor of chlorophytes and streptophytes, we have undertaken the sequencing of Mesostigma mitochondrial DNA (mtDNA). The complete mtDNA sequences of seven chlorophytes previously reported (see following) and two streptophytes (the liverwort Marchantia polymorpha [Oda et al. 1992Citation ] and the angiosperm Arabidopsis thaliana [Unseld et al. 1997Citation ]) have revealed a great diversity of genome organizations and evolutionary patterns, yielding limited information about the nature of the ancestral green algal mitochondrial genome.

The mtDNAs of the prasinophyte Nephroselmis olivacea (Turmel et al. 1999Citation ) and the nonphotosynthetic trebouxiophyte Prototheca wickerhamii (Wolff et al. 1994Citation ) display an evolutionary pattern termed ancestral (Turmel et al. 1999Citation ), that is characterized by a small genome size (45,223 and 55,328 bp, respectively) and a high density of genes. These chlorophyte mtDNAs and their land plant homologs have retained gene clusters that represent vestiges of prokaryotic operon organization. On the other hand, the mtDNAs of four other chlorophytes (Pedinomonas minor [Turmel et al. 1999Citation ], a green alga of uncertain affiliation, and three members of the Chlorophyceae belonging to the order Chlamydomonadales [Boer and Gray 1991Citation ; Vahrenholz et al. 1993Citation ; Denovan-Wright and Lee 1994Citation ; Kroymann and Zetsche 1998Citation ]) display a reduced derived pattern that is characterized by a severe reduction in size (15,758–25,137 bp) and gene number, the presence of only remnants of prokaryotic operons, and the acquisition of several other derived features such as fragmented rRNA genes, an accelerated rate of sequence evolution, a modified genetic code, and a biased codon usage. The 42,919-bp mtDNA of the chlorophycean green alga Scenedesmus obliquus exhibits features of both the ancestral and reduced derived types (Kück, Jekosch, and Holzamer 2000Citation ; Nedelcu et al. 2000Citation ). Unlike the aforementioned green algal mtDNAs, land plant mtDNAs feature an expanded pattern that is marked by an increased size relative to chlorophyte mtDNAs (see table 1 ), the tendency to expand through the incorporation of new sequences in intergenic spacers, the loss of a few genes, as well as the appearance of trans-splicing of group II introns and the acquisition of RNA editing in angiosperms. Given the limited sampling of green plant mtDNAs sequenced to date, it is not clear whether the events that led to the large size of land mtDNAs originated during the evolution of charophytes or before the emergence of the Streptophyta and the Chlorophyta. Evidence for the latter hypothesis would indicate that the trend toward a condensed genome in the Chlorophyta was initiated very early after the appearance of this phylum and became even more radical when late-diverging lineages arose within the Chlorophyceae. The origin of the introns present in green plant mtDNAs is also unknown. Although introns have been found in chlorophyte and land plant mtDNAs, their sporadic distribution does not allow one to predict whether some were inherited vertically from the last common ancestor of all green plants.


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Table 1 Genome Features of Mesostigma mtDNA and Other Green Plant mtDNAsa

 
We report here that phylogenetic analyses of mtDNA-encoded proteins support the notion that Mesostigma represents the earliest branch of green plant evolution. Analysis of Mesostigma mtDNA suggests that the last common ancestor of all green plants possessed a highly compact mitochondrial genome encoding at least 75 genes, some of which possibly contained group I introns. Unlike its homolog in the chloroplast, Mesostigma mtDNA harbors a number of derived characteristics that highlight a distinctive evolutionary pattern within the earliest-diverging green plant lineage.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
Strain and Culture Conditions
An axenic strain of Mesostigma viride (NIES-296) was obtained from the National Institute for Environmental Studies (NIES) (Japan). Cultures were grown at 18°C under alternating 12-h light/12-h dark periods in the medium recommended by the NIES Collection (medium C).

DNA Isolation, Cloning, and Sequencing
As the chloroplast and the mitochondrial genomes of Mesostigma viride were found to copurify in CsCl-bisbenzimide density gradients, their nucleotide sequences were determined in parallel using a plasmid library prepared from an A + T rich fraction. Isolation of this fraction and preparation of the library from 1,500 to 3,000 bp fragments obtained by nebulization were carried out as described previously (Lemieux, Otis, and Turmel 2000)Citation . After hybridization of the clones with the original DNA used for cloning, DNA templates from positive clones were prepared with the QIAprep 8 Miniprep kit (Qiagen). All nucleotide sequences were determined with the PRISM dye terminator cycle sequencing kit (Applied Biosystems) on a DNA sequencer (model 343, Applied Biosystems). T3 and T7 primers as well as oligonucleotides complementary to internal regions of plasmid DNA inserts were used to initiate the sequencing reactions. Genomic regions not represented in the clones analyzed were sequenced from PCR-amplified fragments. Sequences were assembled using AUTOASSEMBLER (Version 1.4.0, Applied BioSystems) and analyzed using the Genetics Computer Group (Madison, WI) software (Version 9.1) package. The protein-coding and rRNA genes were identified by BLAST searches (Altschul et al. 1990Citation ) of the nonredundant database at the National Center for Biotechnology Information. The tRNA genes were found using TRNASCAN-SE (Lowe and Eddy 1997Citation ).

The DNA templates sequenced included RT-PCR products that were generated from total cellular RNA with the SuperScript One-Step RT-PCR System from Life Technologies (Burlington, Ontario, Canada). These analyses were carried out to determine whether the introns in nad3 are trans-spliced at the RNA level.

Phylogenetic Analyses
Mitochondrial genome sequences were retrieved from GenBank: Mesostigma viride, AF353999 (this study); Nephroselmis olivacea, AF110138 (Turmel et al. 1999Citation ); Prototheca wickerhamii, U02970 (Wolff et al. 1994Citation ); Marchantia polymorpha, M68929 (Oda et al. 1992Citation ); Arabidopsis thaliana, Y08501 and Y08502 (Unseld et al. 1997Citation ); Chondrus crispus, Z47547 (Leblanc et al. 1995Citation ); Cyanidioschyzon merolae, D89861 (Ohta, Sato, and Kuroiwa 1998Citation ). The data sets were prepared as follows: the deduced amino acid sequences from individual genes were aligned using CLUSTALW 1.74 (Thompson, Higgins, and Gibson 1994Citation ), these protein alignments concatenated, and the ambiguously aligned regions deleted. Symmetric maximum likelihood distances were computed using PUZZLE 4.0.2 (Strimmer and von Haeseler 1996Citation ), whereas Logdet distances were calculated using SPLITSTREE 2.4 (Huson 1998Citation ). Distance and maximum parsimony trees were constructed using NEIGHBOR and PROTPARS, respectively, in PHYLIP 3.573c (Felsenstein 1995Citation ). The JTT and mtREV24 models with either a uniform rate or gamma-distributed rates (over eight categories) of substitutions across sites were used for the distance analyses. The robustness of distance and maximum parsimony trees was assessed by bootstrap percentages after 100 replications. Maximum likelihood analyses with a uniform rate of substitutions across sites were carried out using PROTML in MOLPHY 2.3b3 (Adachi and Hasegawa 1996Citation ), whereas analyses assuming rate heterogeneity (eight gamma categories) were carried out using the AAML program in PAML (Yang 1997Citation ). Local bootstrap probabilities were estimated by resampling of the estimated log likelihood (RELL) after 10,000 replications (Adachi and Hasegawa 1996Citation ).

Comparisons of Amino Acid Substitution Rates in Different Lineages
For this analysis, we used the data set of 4,139 amino acid positions. To compare the rates of substitution in different lineages, we applied the relative rate test as described by Gu and Li (1992)Citation . The sequences of Porphyra purpurea were used as the outgroup.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
Main Features
The Mesostigma mtDNA sequence assembles as a circle of 42,424 bp (fig. 1 ). In terms of size, A + T content, and gene content, it closely resembles those of chlorophytes displaying an ancestral pattern (table 1 ). Relative to its Nephroselmis homolog, Mesostigma mtDNA is only 3 kbp smaller, has almost the same A + T content, and displays the same number of genes (not counting unique open reading frames [ORFs] and intron ORFs). In the coding regions, we have identified 65 conserved genes, a free-standing ORF (orf174) encoding a protein of 174 amino acids that is not obviously homologous to any known protein, and also four intron ORFs, of which only one has no known homolog in databases (see table 4 ). Together, these coding sequences account for 86.6% of the genome, a value representing the highest density of coding sequences among the mtDNAs of streptophytes and basal chlorophytes examined to date (table 1 ).



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Fig. 1.—Gene map of Mesostigma mtDNA. Coding sequences (filled rectangles) on the outside of the map are transcribed in a clockwise direction; those inside the map are transcribed counterclockwise. Transfer RNA genes are indicated by the one-letter amino acid code followed by the anticodon in parentheses. Seven introns (open rectangles) were identified, four of which contain an ORF (hatched rectangle). The intron sequences bordering the nad3 exons (nad3a, nad3b, and nad3c) are spliced in trans at the RNA level

 

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Table 4 Characteristics of the Introns Found in Mesostigma mtDNA and at Identical Gene Locations in Other Genomes

 
Intergenic spacers in Mesostigma mtDNA range from 2 to 1,103 bp in size, with an average size of 94 bp; only five spacers exceed 226 bp. There are two regions of overlapping genes, rpl6-rps13 and rps10-rps14, with 1 and 20 bp shared between the 3' and 5' coding regions of the genes present in these clusters, respectively. Sequence overlap between these genes has not been reported in any of the other mtDNAs examined to date.

Mesostigma mtDNA contains four group I introns and three group II introns. The presence of both group I and group II introns in a green plant mtDNA is not without precedent as the two classes of introns have been identified in both Marchantia and Scenedesmus mtDNAs.

Conserved Genes
The 65 conserved genes identified in Mesostigma mtDNA encode three rRNA species, 26 tRNAs, 15 ribosomal proteins, 19 components of the respiratory chain and ATP synthase, one component of the Sec-independent protein translocation pathway, and one protein of unknown function (table 2 ). Although none of these genes represents a novel coding sequence among green plant mtDNAs, it is noteworthy that four of them (rps1, sdh3, sdh4, and trnL[caa]) have not been reported previously in chlorophyte mtDNAs and that two others (rpl14 and trnI[gau]) have not been identified in the streptophyte mtDNAs analyzed so far. With 69 conserved genes, Marchantia is the only known green plant that has a larger mitochondrial gene repertoire than Mesostigma. Marchantia mtDNA encodes 27 tRNAs instead of 26 as in Mesostigma mtDNA (the extra tRNAIle[cau] and tRNAThr[ggu], but not tRNAIle[gau]), 16 ribosomal proteins instead of 15 (the extra Rpl2 and Rps8, but not Rpl6), and three components involved in cytochrome c biogenesis (YejR, YejU and YejV), the genes for the last products being absent from Mesostigma mtDNA.


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Table 2 Functions of Conserved Genes in Mesostigma mtDNA

 
The great majority of the conserved genes in Mesostigma mtDNA specify products that appear to be structurally similar to their mitochondrial counterparts in basal chlorophytes and in streptophytes as well as in most protists examined so far. This is the case for the small and large subunit rRNAs (1,558 and 2,843 nt, respectively) whose potential secondary structures approximate those of their equivalents in Escherichia coli (1,542 and 2,904 nt, respectively). All of the tRNAs encoded by Mesostigma mtDNA can also adopt conventional secondary structures, with very few atypical features. Figure 2 shows that Mesostigma mitochondrial 5S rRNA (142 nt long) shares obvious sequence identities and substantial secondary structure similarities with the 5S rRNAs of four other green plants and two red algae. Although its structure is somewhat peculiar in displaying 26–27 extra nucleotides in the variable loop B, it presents no deviation from the universally conserved features of the mitochondrial consensus structure (Lang, Goff, and Gray 1996Citation ; Burger et al. 1999Citation ). Regarding the mitochondrially-encoded proteins of Mesostigma, alignments of their predicted sequences with those of homologs in other mitochondrial lineages revealed that only Rps1 shows a very different structure compared with its counterparts. The predicted sequence of this protein (105 amino acids) appears 76–81 amino acids shorter at its amino-terminus than its counterparts in Marchantia and the heterotrophic jakobid flagellate Reclinomonas americana. As the corresponding extensions in the latter organisms can be unambiguously aligned (with only one gap of a single amino acid), it is possible that Rps1 is not a functional protein in Mesostigma.



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Fig. 2.—Comparative analysis of mitochondrially encoded 5S rRNAs from Mesostigma, four other green plants, and two red algae. A, Potential secondary structure of Mesostigma mitochondrial 5S rRNA. Helices (I–V) and loops (A–E) are denoted as in Moore (1995)Citation . Nonstandard pairings are highlighted by small filled (purine-pyrimidine), small open (purine-purine), and large filled (pyrimidine-pyrimidine) circles. B, Alignment of Mesostigma mitochondrial 5S rRNA with its homologs in other green plants and red algae. The alignment is based on secondary structure models of the compared rRNAs. Residues identical in five or more of the taxa are indicated by white letters on a black background. Dashes denote gaps in the alignment. Horizontal lines above the alignment denote the regions that are part of helices in the secondary structures. All 5S rRNA sequences were inferred from the corresponding DNA sequences. For the GenBank accession numbers of these nucleotide sequences, refer to Materials and Methods. MESOvirid, Mesostigma viride; NEPHoliva, Nephroselmis olivacea; PROTwicke, Prototheca wickerhamii; MARCpolym, Marchantia polymorpha; ARABthali, Arabidopsis thaliana; CHONcrisp, Chondrus crispus; CYANmerol, Cyanidioschyzon merolae. The sequence of Ch. crispus 5S rRNA was taken from Gray et al. (1998)Citation

 
Genetic Code and Codon Usage
Like all of its green plant counterparts whose genomes have been sequenced so far, with two exceptions (Scenedesmus and Pedinomonas mitochondria), the Mesostigma mitochondrion uses the standard genetic code. Table 3 presents the codon usage in the 36 conserved mitochondrial protein-coding genes of Mesostigma. All 61 sense codons are used, although there is a strong bias for the codons that end in A or U in four-codon families. The UAA termination codon is found in all conserved genes, except rps1, in which UAG is present.


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Table 3 Codon Usage in the 36 Conserved Mitochondrial Protein-coding Genes of Mesostigma

 
Whereas the 26 tRNAs encoded by Mesostigma mtDNA are not sufficient to decode all of the sense codons that occur in this green plant mtDNA, some appear to be redundant. A minimum of two tRNA genes are missing from Mesostigma mtDNA, namely trnT(ugu) for translating the ACN codons, and trnI(cau) for decoding AUA (table 3 ). A trnT(ugu) gene is also absent from several of the mtDNAs investigated so far, including red algal mtDNAs; moreover, the absence of a mitochondrially encoded trnI(cau) gene has been reported in the latter red algae and the derived chlorophytes Pedinomonas and Chlamydomonas (Gray et al. 1998Citation ). Prototheca mtDNA is the only known green plant mtDNA that contains trnT(ugu) and encodes a complete set of tRNAs for complete translation of all mitochondrial codons. The tRNAs encoded by the missing Mesostigma genes are presumably imported from the cytosol. Given that tRNAArg(ucg) has a U residue in the wobble position and thus can presumably decode all four CGN arginine codons, the presence of trnR(acg) in Mesostigma mtDNA is functionally redundant (table 3 ). Note here that, in the tRNA specified by the latter gene, the A residue in the wobble position is assumed to be converted to inosine posttranscriptionally, potentially allowing the tRNA to also decode all four CGN codons (Pfitzinger et al. 1990Citation ). Also redundant are trnL(caa) and trnG(gcc) because tRNALeu(uaa) is available to read both the TTA and TTG codons, and tRNAGly(ucc) can decode all four GGN glycine codons.

Gene Organization
All the genes in the 18,114-bp region of Mesostigma mtDNA which is delimited by K(uuu) and rns, with the exception of two unlinked tRNA genes, are encoded by the same strand, whereas all the genes in the rest of the genome, except again tRNA genes (five individual ones and one pair), are located on the complementary strand (fig. 1 ). This gene distribution predicts a minimum of 16 potential transcription units, seven of which are monocistronic and made up of tRNA genes. In Nephroselmis mtDNA, potential transcription units are also numerous, but are predicted to be mainly multicistronic. Genes in Prototheca mtDNA form as few as two potential transcription units.

Comparison of gene orders in Mesostigma mtDNA and other mtDNAs reveals only two conserved gene clusters, rps12-rps7 and rpl6-rps13. Commonly found in protist mtDNAs, these gene pairs are also present in the genomes of the bacterial progenitors of mitochondria in which they are part of the large ribosomal gene cluster corresponding to the str, S10, spc, and {alpha} operons of E. coli (Lang, Gray, and Burger 1999Citation ). Of the green plant mtDNAs examined so far, that of Nephroselmis has retained the largest fraction of this ancestral gene cluster.

Intron Content
Table 4 summarizes the features of the seven introns found in Mesostigma mtDNA. Two group I introns are located in rnl (Mvrnl·1 and Mvrnl·2), whereas two others lie in cox1 (Mvcox1·1 and Mvcox1·2). We have classified these four group I introns within distinct subdivisions of the group I family (fig. 3 ) on the basis of their sequences and secondary structures (Michel and Westhof 1990Citation ). Each of the rnl introns as well as Mvcox1·2 contains an ORF specifying a potential homing endonuclease of the LAGLIDADG family. Experimental evidence for such an enzymatic activity has been demonstrated for the homologous proteins encoded by chloroplast group I introns residing at the same insertion sites as Mvrnl·1 and Mvrnl·2 within the LSU rRNA gene (Dürrenberger and Rochaix 1991Citation ; Turmel et al. 1995Citation ; Lucas et al. 2001Citation ). Apart from the introns in the mtDNA of the lobose amoeba Acanthamoeba castellanii and the bacterium Simkania negevensis, introns occupying the same positions as the Mesostigma mitochondrial rnl introns have been reported only in green algae (table 4 ). Introns residing at the same positions as Mvcox1·1 and Mvcox1·2 have also been described previously (table 4 ). Of the four group I insertion sites in Mesostigma mtDNA, that occupied by Mvcox1·2 features the broadest phylogenetic distribution; introns at this site have been found in the mtDNA of various yeasts and fungi, the slime mold Dictyostelium discoideum, the green alga Chlamydomonas eugametos, and the liverwort Marchantia. Each of these coxI introns, except that of Allomyces macrogynus, displays an ORF; however, unlike the situation that prevails for the other introns sharing insertion sites with Mesostigma rnl introns, the ORF is found at more than one location within the intron sequences (L1 and L9.1), and unrelated ORFs occupy the different positions.



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Fig. 3.—Structural similarities between the group I introns in Mesostigma mtDNA and their closest homologs in other genomes. Mvrnl·1 and Mvrnl·2 were compared with the homologous rnl introns in the chromosomal DNA of Simkania and in Acanthamoeba mtDNA, respectively, whereas Mvcox1·1 and Mvcox1·2 were compared with the homologous introns in Podospora mtDNA. Identical residues are represented in uppercase bold and italic characters; nonconserved residues are indicated in lowercase. Splice sites between the exon and intron residues are denoted by arrows. Introns are modelled according to the nomenclature proposed by Burke et al. (1987)Citation

 
As revealed by comparative analyses of the Mesostigma mitochondrial group I introns and their homologs, most, if not all, of the introns sharing common insertion sites are closely related. They display not only similar sequences and structures but also similar ORFs when the latter are identically positioned. Phylogenetic analyses did not allow us to trace the precise origin of the Mesostigma mitochondrial group I introns because statistical support for several nodes was weak. Interestingly, these analyses as well as comparative analyses of the ORF sequences revealed that the closest homologs of the Mesostigma introns originate from nongreen plants. The introns most closely related to Mvrnl·1 and Mvrnl·2 are those found in the chromosomal DNA of Simkania and in Acanthamoeba mtDNA, whereas the introns most closely resembling Mvcox1·1 and Mvcox1·2 are the homologs in Podospora mtDNA (fig. 3 ). These results suggest that some of the group I introns analyzed were acquired by lateral transfer. In particular, the aforementioned Simkania rnl intron is likely to have been acquired by lateral transfer of an intron sequence from the mitochondrial genome of an Acanthamoeba-like organism, as Simkania is an obligate endosymbiont and a close relative of Parachlamydia acanthamoebae, an intracellular parasite of Acanthamoeba (Everett, Bush, and Andersen 1999Citation ). While we reported evidence suggesting that the Acanthamoeba mitochondrial introns inserted at the same positions as Mvrnl·1 and Mvrnl·2 were acquired by lateral transfer of green algal chloroplast introns (Turmel et al. 1995Citation ), the more extensive data now available on intron distributions in the rnl gene indicate that these Acanthamoeba introns might have arisen by lateral transfer of green algal mitochondrial introns. Alternatively, considering the evidence that Mesostigma appeared before the divergence of streptophytes and chlorophytes (Lemieux, Otis, and Turmel 2000Citation ; also see following) and also the possibility that Acanthamoeba and green plants share a common ancestor (Baldauf et al. 2000Citation ), the Mesostigma rnl introns and their Acanthamoeba homologs might have been inherited vertically from a common ancestor.

The insertion sites of the three group II introns in Mesostigma mtDNA are unique to this mitochondrial genome. Two introns reside in nad3 (Mvnad3·1 and Mvnad3·2) and the third in cox2 (Mvcox2·1). All three introns belong to the subdivision IIA. As they differ from the majority of group II introns characterized previously (Michel and Ferat 1995Citation ) by their degenerate domain I (fig. 4 ), they probably require factors for splicing. Mvnad3·2 also lacks domain II, a region dispensable for intron splicing (Michel and Ferat 1995Citation ) but almost universally present in group II introns (fig. 4 ). The two Mesostigma nad3 introns are bipartite: the two pieces constituting each of these introns are far apart on the mitochondrial genome and occur in separate transcription units (fig. 1 ). Both introns are trans-spliced at the RNA level in vivo, as demonstrated by sequencing nad3 cDNAs encompassing each or both of the intron insertion sites. The breakpoint of each intron lies within domain IV (fig. 4 ), a common feature of trans-spliced group II introns (Michel and Ferat 1995Citation ). The two Mesostigma nad3 introns lack an ORF, whereas Mvcox2·1 encodes a potential protein. Unlike most group II intron–encoded proteins identified so far (Michel and Ferat 1995Citation ), this protein does not exhibit a polymerase domain of reverse transcriptases; it shares only very limited sequence identity with other group II intron–encoded proteins.



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Fig. 4.—Secondary structure models of the group II introns in Mesostigma mtDNA. Intron modelling was according to the nomenclature proposed for group II introns (Michel, Umesono, and Ozeki 1989Citation ). Roman numbers specify the major structural domains. Tertiary interactions are denoted by dashed lines, curved arrows, or Greek lettering. EBS and IBS are exon-binding and intron-binding sites, respectively. Nucleotides that potentially participate in the {delta}-{delta}' interaction are boxed. Arrowheads denote intron-exon junctions. The nucleotides shown in lowercase letters in domain IV of Mvnad3·2 are ymf16 coding sequences. Note that the precise positions of the breakpoints of domain IV in the two nad3 introns are unknown

 
Phylogenetic Analyses
The protein sequences (4,139 amino acid positions) derived from 19 genes that are common to the mtDNAs of Mesostigma, Marchantia, Arabidopsis, Nephroselmis and Prototheca were concatenated and analyzed with various phylogenetic inference methods using as outgroups the homologous sequences from three red algae (Porphyra purpurea, Chondrus crispus and Cyanidioschyzon merolae). The complete mtDNA sequences of Pedinomonas and the four chlorophycean green algae investigated so far were not used because of the long-branch attraction artifacts caused by their considerably higher rate of evolution relative to the selected green algal sequences (Turmel et al. 1999Citation ; Nedelcu et al. 2000Citation ). The JTT model of amino acid replacement was selected for the distance and maximum likelihood analyses because it gave higher likelihood values than the mtREV24 model in PROTML analyses. As shown in figure 5 , trees constructed with distance, maximum parsimony, and maximum likelihood methods using a uniform rate of substitution across sites are congruent in showing a strongly supported topology in which Mesostigma emerges before the divergence of the Streptophyta and the Chlorophyta. In maximum likelihood analyses, the topology of the best tree (fig. 5 ) accounted for 83.3% of the RELL bootstrap samples, and two of the five alternative topologies detected (representing 12.9% of the RELL samples) also revealed Mesostigma as the earliest divergence among green plants. In one of the alternative topologies (0.4% of the RELL samples), Mesostigma was found at the base of the land plants within the Streptophyta, and in the two remaining topologies (3.2% of the RELL samples), its position was also basal relative to land plants, with Nephroselmis and Prototheca representing earlier diverging lineages. When the Shimodaira-Hasegawa test (Shimodaira and Hasegawa 1999Citation ) was used to compare the topology of the best tree with those of the 14 other possible trees in which Marchantia and Arabidopsis were constrained to be paired, the best tree was not found to be significantly different (P < 0.05) from those affiliating Mesostigma with land plants.



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Fig. 5.—Phylogenetic position of Mesostigma as inferred from the deduced amino acid sequences (4,139 positions) from 19 mitochondrial genes. The best tree computed with PROTML is shown. Bootstrap values obtained in PROTML, distance, and maximum parsimony analyses assuming a uniform rate of substitution across sites are indicated above the nodes at the upper, middle, and lower positions, respectively. Following are the names of the 19 genes, with lengths of alignment used indicated in parentheses: atpA (229), atp8 (35), atp9 (71), cob (359), cox1 (489), cox2 (223), cox3 (259), nad1 (316), nad2 (380), nad3 (104), nad4 (447), nad4L (100), nad5 (571), nad6 (145), rpl16 (74), rps3 (82), rps12 (114), ymf16 (106), and ymf39 (35)

 
As incorrect tree topologies can be recovered when invariable sites and rate variation across sites are not taken into account (Lockhart et al. 1996Citation ; Silberman et al. 1999Citation ; Stiller and Hall 1999Citation ), we tested the effect of these two parameters on the phylogenetic analyses presented in figure 5 . The distance trees recovered after considering rate heterogeneity or removing all 1,559 constant sites in the data set displayed a topology identical to that shown in figure 5 , and levels of support for the various nodes remained at 100% (table 5 ). Similarly, in maximum likelihood analyses, we observed no change in the topology of the best tree, but the levels of support for Mesostigma being the earliest green plant lineage and for monophyly of the chlorophytes were found to be lower (table 5 ). In the Shimodaira-Hasegawa test, no significant difference (P < 0.05) was noted between the best tree and those identifying Mesostigma as a basal lineage within the Streptophyta.


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Table 5 Effect of Inference Methods on Support for Two Nodes in Tree of Figure 5

 
As incorrect relationships in reconstructed trees can also be caused by systematic biases in amino acid composition (Lockhart et al. 1999Citation ), we also tested whether this factor influenced our phylogenetic conclusions. Chi-square analysis of amino acid compositions at variable sites revealed that the 19 concatenated proteins of Mesostigma do not differ significantly (P < 0.005) from those of Nephroselmis, Prototheca, and Marchantia. Our failure to detect a systematic bias in amino acid composition within green plants suggests that the position inferred for Mesostigma in our phylogenetic analyses is the result of a genuine phylogenetic signal. Consistent with this conclusion, neighbor-joining analysis of LogDet distances calculated after removal of all constant sites placed Mesostigma as the earliest diverging green plant lineage in 74% of the bootstrap samples (table 5 ). LogDet distances allow the recovery of the correct tree when sequences differ markedly in amino acid frequencies for cases where substitution processes are otherwise uniform across the underlying tree (Lockhart et al. 1994Citation ).

Rates of Amino Acid Substitutions in Different Lineages
The branch lengths in the maximum likelihood tree shown in figure 5 suggest that Mesostigma mtDNA has evolved at a rate similar to those observed for the mtDNAs of Nephroselmis, Prototheca and Arabidopsis. To compare more precisely the rates of amino acid substitution in these green plant lineages, we used the method developed by Gu and Li (1992)Citation to demonstrate a higher rate of sequence evolution in rodents than in humans. When we compared the rate of amino acid substitution in the lineage leading to Mesostigma with those leading to other green plants (table 6 ), only the Mesostigma and Nephroselmis lineages showed a significant difference at P < 0.001, thus providing evidence for an overall faster substitution rate in the lineage leading to Mesostigma relative to the lineage leading to Nephroselmis (table 6 ). At P < 0.05, the rate observed for the Mesostigma lineage significantly differed from the rates exhibited by all other lineages examined, except the Prototheca lineage. Comparisons of other pairs of green plant lineages showed that amino acid substitution rate is significantly (P < 0.001) faster in the lineage leading to Arabidopsis than in the Marchantia and Prototheca lineages. Note here that the amino acid differences observed for the pairs of green plants, including Arabidopsis, were overestimated, as the large number of edited sites in mRNA derived from Arabidopsis mitochondrial genes (Giege and Brennicke 1999Citation ) were not taken into account in our analysis.


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Table 6 Differences in the Number of Amino Acid Substitutions in 19 Mitochondrial Proteins and Test for Equal Rates of Substitutions in Selected Pairs of Green Plant Lineages

 
Influence of Outgroup on the Phylogenetic Position of Mesostigma
The previous comparisons of amino acid substitution rates suggest the possibility that our placement of Mesostigma at the base of the other green plants is a long-branch attraction artifact resulting from the inclusion of the red algal outgroup taxa. Not supporting this hypothesis is our finding that the branching order shown in figure 5 for the five ingroup taxa was still the preferred topology when we analyzed with maximum likelihood under gamma-distributed rates the three possible unrooted four-taxon trees in which the streptophytes Marchantia and Arabidopsis were constrained to be paired. This topology was recovered in 96.9% of the RELL bootstrap samples; the unrooted topology in which Mesostigma is sister to Prototheca represented the rest of the RELL bootstrap samples. In the Shimodaira-Hasegawa test, the best tree proved to be significantly different (P < 0.05) from the two alternative trees.


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
Our phylogenetic analyses of concatenated mtDNA-encoded protein sequences are congruent with our previous analyses of cpDNA-encoded rRNAs and proteins (Lemieux, Otis, and Turmel 2000Citation ) in favoring the hypothesis that Mesostigma represents a lineage that appeared before the divergence of the Streptophyta and the Chlorophyta. Although all analyses of the mitochondrial data set favored this placement, support in some of the analyses was weaker than those in comparable analyses of the chloroplast data set. We observed decreased support not only for the basal position of Mesostigma but also for the monophyly of chlorophytes (table 5 ), suggesting that the phylogenetic signal provided by the data set of 19 mtDNA-encoded proteins is not sufficient to resolve unambiguously the divergence order of deeply branching green plant lineages. When more mitochondrial genome sequences of green plant taxa with an expanded or ancestral pattern of evolution become available, it will be important to test if inclusion of these taxa in phylogenetic analyses affects the position of Mesostigma. Increasing the number of taxa sampled per lineage is believed to yield more accurate trees (Graybeal 1998Citation ).

Comparison of the gene content and the gene organization of Mesostigma mtDNA with those of other green plant mtDNAs disclosed only a few ancestral features that support the positioning of Mesostigma as the earliest branch of green plant evolution. The most important of these structural features is the presence in Mesostigma mtDNA of two genes that are specific to chlorophytes (rpl14 and trnI[gau]) and of four genes that are specific to streptophytes (rps1, sdh3, sd4, trnL[caa]) (fig. 6 ). The high density of genes encoded by Mesostigma mtDNA is also consistent with the notion that this prasinophyte represents the earliest green plant divergence (see following). Both the aforementioned features have been identified in the chloroplast genome of Mesostigma (Lemieux, Otis, and Turmel 2000Citation ). This genome exhibits the highest gene density among the green plant cpDNAs investigated so far. Thirteen of its 135 genes have been found to be specific to chlorophytes and seven to streptophytes.



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Fig. 6.—Phylogenetic distribution of mitochondrial gene loss in the Chlorophyta and the Streptophyta. Loss events were mapped on the tree shown in figure 5 . Numbers below species names indicate the number of genes in the corresponding mtDNA. Note that intron-encoded genes were not considered here. Genes that were lost independently in different lineages are underlined

 
Clearly, the mitochondrial genome of Mesostigma has retained fewer ancestral features than the chloroplast genome. The gene content of the green plant mtDNAs sequenced so far predicts that a minimum of 75 genes were present in the mitochondrial genome of the common ancestor of all green plants; 10 of these 75 genes were specifically lost in the lineage leading to Mesostigma (fig. 6 ). Gene loss occurred at a lower rate in the chloroplast genome; only six of the 141 genes that are predicted to be encoded by the cpDNA of the common ancestor of all green plants were specifically lost in the Mesostigma lineage (Lemieux, Otis, and Turmel 2000Citation ). The high rate of gene loss in the mitochondrial genome may account for the total absence of genes specifically shared between Mesostigma and nongreen algal mtDNAs. Five genes in Mesostigma cpDNA are specifically shared with the Synechocystis cyanobacterial genome or the chloroplast genomes of the red algae Porphyra and Cyanidium, further supporting the idea that Mesostigma occupies the most basal position among the green plants analyzed so far (Lemieux, Otis, and Turmel 2000Citation ). With regard to gene organization, the study reported here reveals that none of the ancestral operons identified in Nephroselmis mtDNA has remained intact in Mesostigma mtDNA. In sharp contrast, all of the operons found in chlorophyte and streptophyte cpDNAs have been preserved in the chloroplast genome of Mesostigma (Lemieux, Otis, and Turmel 2000Citation ). In fact, gene order has been so highly conserved during the evolution of streptophyte cpDNAs that many gene clusters are still shared between land plant and Mesostigma cpDNAs. Finally, some of the introns in Mesostigma mtDNA reflect the loss of ancestral gene structures (i.e., of continuous coding sequences), as their insertion in the mitochondrial genome most probably took place by lateral transfer after the emergence of the Mesostigma lineage (see following). Like its red algal counterparts, Mesostigma cpDNA harbors no introns, a condition considered to be ancestral (Lemieux, Otis, and Turmel 2000Citation ).

Of the three types of evolutionary patterns that have been attributed to green plant mtDNAs (Turmel et al. 1999Citation ), the ancestral pattern best describes the evolutionary trends displayed by Mesostigma mtDNA. At both the size and gene density levels, Mesostigma mtDNA most closely resembles the two ancestral mtDNAs that have been sequenced to date (Nephroselmis and Prototheca mtDNAs); however, unlike these mtDNAs, it exhibits virtually no vestiges of prokaryotic gene organization. Although Mesostigma mtDNA shows a number of derived organizational features, its evolutionary pattern is distinct from that of Scenedesmus mtDNA and thus cannot be considered as intermediate between the ancestral and reduced derived patterns. Compared with Scenedesmus mtDNA, Mesostigma mtDNA diplays a substantially higher gene density (86.6% vs. 60.6%) and evolves much more slowly at the sequence level. As revealed by our analysis of amino acid substitutions in 19 mtDNA-encoded proteins, the rate of sequence evolution of Mesostigma mtDNA is comparable to that of Prototheca mtDNA but is slightly higher than that of Nephroselmis mtDNA (table 6 ).

Given the irregular distribution and poor phylogenetic content of the introns identified to date in green plant mtDNAs (Gray et al. 1998Citation ), inferring the intron makeup of the mitochondrial genome in the common ancestor of all green plants is a highly speculative endeavor. It appears that only two of the seven introns in Mesostigma mtDNA, the introns in the rnl gene, might have been inherited from the common ancestor of all green plants. The three group II introns in Mesostigma mtDNA are unlikely to have been acquired by vertical descent from this common ancestor because their insertion sites are unique to Mesostigma mtDNA. Although their origin remains unclear, they most probably took residence in this mtDNA via lateral transfer. Considering that all three introns belong to the same subgroup and share a degenerate domain I, it seems plausible that they arose following lateral transfer of a group II intron and subsequent transposition of this intron at two other mtDNA loci. In contrast, all four group I introns in Mesostigma mtDNA have a number of close relatives at identical gene locations in other genomes. Because the homologs of the two Mesostigma cox1 introns are prevalent in yeast and fungal mtDNAs and occur sporadically in green plant mtDNAs (table 4 ), both these introns were probably acquired following lateral transfer events between the mitochondria of divergent organisms.

The notion that the mitochondrial rnl introns of Mesostigma were inherited vertically from the common ancestor of all green plants is consistent with the finding that their homologs occupy multiple green plant lineages (including deep branches) and also with their limited occurrence outside this phylum. Their nongreen plant homologs are restricted to Acanthamoeba mtDNA and to the genome of the bacterium Simkania (see Results for a discussion on the possible origin of these introns). Considering that the mobility of the two Mesostigma rnl introns and their homologs is facilitated by intron-encoded homing endonucleases (Lucas et al. 2001Citation ), it is surprising that these introns are not more widespread among eukaryotes. The reason for such a limited phylogenetic distribution is not obvious.

Relatively early during the evolution of green algae, homologs of the Mesostigma mitochondrial rnl introns appear to have been successful in invading the chloroplast genome and establishing a niche at cognate sites in the rnl gene. This transfer of genetic information might have occurred between the mitochondria and chloroplast of the same cell, as DNA exchange between these organelles has been documented (Palmer 1985Citation ). Interorganellar transfer of rnl introns is supported by the finding that homologs of the two Mesostigma rnl introns have been identified not only in mtDNA but also in cpDNA (table 4 ). In some green algae such as Monomastix, introns similar to their Mesostigma homologs have been found in both organelle genomes (Lucas et al. 2001Citation ). We favor the view that rnl introns were transferred from mitochondria to the chloroplast, as absence of introns is an ancestral character for the chloroplast genome (both Mesostigma and Nephroselmis harbor introns in their mitochondrial genome but none in their chloroplast genome).

We have shown that the two group II introns in the Mesostigma nad3 gene feature a bipartite structure and are trans-spliced at the RNA level in vivo. Prior to our study, trans-splicing of mitochondrial group II introns had been described only in land plants, more specifically in angiosperms (Michel and Ferat 1995Citation ). It is very unlikely that trans-spliced group II introns were present in the mitochondrial genome of the common ancestor of green plants, as three lines of evidence support the idea that trans-splicing of group II introns originated independently in Mesostigma and streptophyte mtDNAs. First, the mitochondrial genome of the bryophyte Marchantia lacks any trans-spliced introns, suggesting that trans-splicing appeared late during the evolution of streptophytes (after the divergence of bryophytes and vascular plants). Second, no introns have been identified in the nad3 gene of land plants. Third, the trans-spliced introns found in Mesostigma and angiosperm mtDNAs differ remarkably in structure, notably with respect to domain I. Trans-splicing in both the Mesostigma and streptophyte lineages likely arose following genome rearrangements that led to fragmentation of cis-spliced introns and dispersal of the resulting pieces (Michel and Ferat 1995Citation ).

In conclusion, 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 at least 75 genes and perhaps two group I introns in the rnl gene. Mesostigma features the most compact mtDNA among the green plants analyzed so far. Considering that land plant mtDNAs are much larger in size and continue to expand in size because of the incorporation of foreign sequences (Palmer 1985Citation ), we infer that mtDNA size began to increase dramatically in the Streptophyta, either during the evolution of charophytes or during the transition of the charophytes to land plants. Complete sequencing of mtDNAs from a number of charophytes and early-diverging land plants will be required to distinguish between these two hypotheses.


    Supplementary Material
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
The genome sequence reported in this paper has been deposited in the GenBank database (accession number AF353999).


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
We thank Dr. Charles Delwiche and an anonymous reviewer for their constructive criticisms. M.T. and C.L. are Associates in the Program in Evolutionary Biology of the Canadian Institute for Advanced Research. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (GP0003293 to M.T. and GP0002830 to C.L. and M.T.).


    Footnotes
 
Geoffrey McFadden, Reviewing Editor

Abbreviations: cpDNA, chloroplast DNA; mtDNA, mitochondrial DNA; NIES, National Institute for Environmental Studies; ORF, open reading frame; RELL, resampling of the estimated log likelihood; rRNAs, ribosomal RNAs. Back

Keywords: green algae Mesostigma viride mitochondrial DNA group I introns group II introns trans-splicing Back

Address for correspondence and reprints: Monique Turmel, Département de Biochimie et de Microbiologie, Pavillon C.-E. Marchand, Université Laval, Québec (QC) G1K 7P4, Canada. monique.turmel{at}rsvs.ulaval.ca . Back


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Accepted for publication August 21, 2001.