The Complete Mitochondrial DNA Sequence of the Green Alga Pseudendoclonium akinetum (Ulvophyceae) Highlights Distinctive Evolutionary Trends
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分子生物学进展 2004年第5期
Département de Biochimie et de Microbiologie, Université Laval, Québec, Québec, Canada
E-mail: Monique.Turmel@rsvs.ulaval.ca.
Abstract
The mitochondrial genome has undergone radical changes in both the Chlorophyta and Streptophyta, yet little is known about the dynamics of mtDNA evolution in either of these lineages. In the Chlorophyta, which comprises four of the five recognized classes of green algae (Prasinophyceae, Trebouxiophyceae, Ulvophyceae, and Chlorophyceae), the mitochondrial genome varies from 16 to 55 kb. This genome has retained a compact gene organization and a relatively complex gene repertoire ("ancestral" pattern) in the basal lineages represented by the Trebouxiophyceae and Prasinophyceae, whereas it has been reduced in size and gene complement and tends to evolve much more rapidly at the sequence level ("reduced-derived" pattern of evolution) in the Chlorophyceae and the lineage leading to the enigmatic chlorophyte Pedinomonas. To gain information about the evolutionary trends of mtDNA in the Ulvophyceae and also to gain insights into the phylogenetic relationships between ulvophytes and other chlorophytes, we have determined the mtDNA sequence of Pseudendoclonium akinetum. At 95,880 bp, Pseudendoclonium mtDNA is the largest green-algal mitochondrial genome sequenced to date and has the lowest gene density. These derived features are reminiscent of the "expanded" pattern exhibited by embryophyte mtDNAs, indicating that convergent evolution towards genome expansion has occurred independently in the Chlorophyta and Streptophyta. With 57 conserved genes, the gene repertoire of Pseudendoclonium mtDNA is slightly smaller than those of the prasinophyte Nephroselmis olivacea and the trebouxiophyte Prototheca wickerhamii. This ulvophyte mtDNA contains seven group I introns, four of which have homologs in green-algal mtDNAs displaying an "ancestral" or a "reduced-derived" pattern of evolution. Like its counterpart in the chlorophycean green alga Scenedesmus obliquus, it features numerous small, dispersed repeats in intergenic regions and introns. Its overall rate of sequence evolution appears to be accelerated to an intermediary level as compared with the rates observed in "ancestral" and "reduced-derived" mtDNAs. In agreement with the finding that Pseudendoclonium mtDNA exhibits features typical of both the "ancestral" and "reduced-derived" patterns of evolution, phylogenetic analyses of seven mtDNA-encoded proteins revealed a sister-group relationship between this ulvophyte and chlorophytes displaying "reduced-derived" mtDNAs.
Key Words: Green algae ? Ulvophyceae ? Pseudendoclonium akinetum ? mitochondrial DNA ? group I introns ? repeated sequences
Introduction
The mitochondrial genomes of green plants are highly variable in size, gene content, and organization and show divergent evolutionary trends in some lineages, as revealed by the complete mtDNA sequences that have been reported thus far for 13 members of this group. Representatives of the two main phyla of green plants, the Streptophyta and Chlorophyta, have been examined in these genome analyses. The Streptophyta (Bremer 1985) comprises all embryophytes (land plants) and their closest green-algal relatives, the members of the class Charophyceae sensu Mattox and Stewart (1984). In this phylum, the mitochondrial genomes of the bryophyte Marchantia polymorpha (Oda et al. 1992), of three angiosperms (Arabidopsis thaliana [Unseld et al. 1997], Beta vulgaris [Kubo et al. 2000], and Oryza sativa [Notsu et al. 2002]), and of the charophycean green alga Chaetosphaeridium globosum (Turmel, Otis, and Lemieux 2002b) have been entirely sequenced. The Chlorophyta (Sluiman 1985) comprises the four other classes of green algae: the Prasinophyceae, Ulvophyceae, Trebouxiophyceae, and Chlorophyceae. The seven chlorophyte mitochondrial DNA (mtDNA) sequences that are currently available include those of four chlorophycean green algae (Chlamydomonas reinhardtii [Michaelis, Vahrenholz, and Pratje 1990], Chlamydomonas eugametos [Denovan-Wright, Nedelcu, and Lee 1998], Chlorogonium elongatum [Kroymann and Zetsche 1998], and Scenedesmus obliquus [Kück, Jekosch, and Holzamer 2000; Nedelcu et al. 2000]), the nonphotosynthetic trebouxiophyte Prototheca wickerhamii (Wolff et al. 1994), the prasinophyte Nephroselmis olivacea (Turmel et al. 1999), and a chlorophyte of uncertain phylogenetic affinity, Pedinomonas minor (Turmel et al. 1999). The remaining green alga whose mitochondrial genome has been scrutinized, Mesostigma viride (Turmel, Otis, and Lemieux 2002a), belongs to the Streptophyta (Bhattacharya et al. 1998; Marin and Melkonian 1999; Karol et al. 2001) or to a lineage that emerged before the divergence of the Streptophyta and Chlorophyta (Lemieux, Otis, and Turmel 2000; Turmel et al. 2002; Turmel, Otis, and Lemieux 2002a).
Because of their large size and their tendency to incorporate foreign DNA (from the nucleus and the chloroplast), land-plant mitochondrial genomes have been reported to feature an "expanded" pattern of evolution (Turmel et al. 1999). These mitochondrial genomes are the largest (187 kb in Marchantia to 2,000 kb in muskmelon) among green plants, and they also show the greatest structural complexity. Most of the increased size of land-plant mtDNAs relative to their green-algal homologs is accounted for by noncoding sequences that reside either in intergenic regions or introns (Oda et al. 1992; Unseld et al. 1997; Kubo et al. 2000; Notsu et al. 2002). Sixty-nine mitochondrial genes have been identified in Marchantia (Oda et al. 1992; Gray et al. 1998), whereas about 50 have been found in angiosperms (Unseld et al. 1997; Kubo et al. 2000; Notsu et al. 2002). This substantial difference in coding capacity is attributed to gene transfer to the nucleus, a widespread and ongoing phenomenon (Schuster and Brennicke 1994; Palmer et al. 2000; Adams et al. 2002). In embryophyte mitochondria, unicircular genome-sized molecules coexist in a dynamic equilibrium with subgenomic circles (Palmer and Shields 1984; Mackenzie, He, and Lyznik 1994). In Marchantia mitochondria, unicircular-genome sized molecules apparently coexist with linear molecules and complex branched structures of multigenomic sizes (Oldenburg and Bendich 2001). In contrast to their fluid structure, land-plant mitochondrial genomes evolve extremely slowly at the sequence level; in angiosperm mitochondria, point mutations can occur at a frequency up to 100 times lower than in vertebrate mitochondria (Wolfe, Li, and Sharp 1987; Palmer and Herbon 1988).
The more compact green-algal mitochondrial genomes display distinctive evolutionary patterns. They range in size from 16 kb (in C. reinhardtii) to 67.8 kb (in Mesostigma) and encode 12 (in C. reinhardtii, C. eugametos, and Chlorogonium) to 67 (in Chaetosphaeridium) genes. An "ancestral" (minimally derived) evolutionary pattern (Turmel et al. 1999) has been ascribed to the circular-mapping mtDNAs of Mesostigma, Chaetosphaeridium, Nephroselmis, and Prototheca, because of their large number of conserved genes (>60), their high gene density, and their important sequence conservation. Not only fewer genes but also a greater variability in gene content (12 to 42 genes) and structural organization (linear or circular-mapping molecules, or even multimeric molecules, as reported for Polytomella [Fan and Lee 2002]) have been found in chlorophycean green-algal mtDNAs. The coding sequences of these genomes are highly divergent from those of other green plants and feature numerous deletions/additions; moreover, the rRNA gene-coding regions are fragmented into pieces that are dispersed throughout the genomes. As a consequence of this high sequence divergence, chlorophycean taxa exhibit very long branches in mitochondrial trees, and, most probably because of long-branch attraction artifacts, usually lie outside the green-plant clade when other green plants and non–green-plant taxa are included in the analyses. A "reduced-derived pattern" (Turmel et al. 1999) of evolution has been assigned to the three smallest and gene-poorest chlorophycean mtDNAs (i.e., to C. reinhardtii, C. eugametos, and Chlorogonium mtDNAs). Because of its less-derived characters, the Scenedesmus mtDNA sequence has been considered to display an "intermediate" pattern of evolution (Nedelcu et al. 2000).
The present study was undertaken to gather information about the evolutionary trends of the mitochondrial genome in the Ulvophyceae and also to gain insights into the phylogenetic relationships between ulvophytes and other chlorophytes. Various hypotheses have been proposed concerning the phylogenetic position of the Ulvophyceae within the Chlorophyta, but none is strongly supported by statistical analyses. On the basis of ultrastructural studies (Mattox and Stewart 1984; O'Kelly and Floyd 1984) and some phylogenetic analyses of nuclear small subunit (SSU) rDNA sequences (Friedl 1995; Bhattacharya, Friedl, and Damberger 1996; Nakayama, Watanabe, and Inouye 1996; Chapman et al. 1998; Watanabe et al. 2000; Wolf et al. 2002), it has been proposed that the Ulvophyceae are a monophyletic assemblage that emerged before the divergence of the Trebouxiophyceae and Chlorophyceae. Independent inferences from nuclear SSU rDNA sequences (Friedl 1997; Marin and Melkonian 1999) and from concatenated chloroplast SSU and large subunit (LSU) (Turmel et al. 2002) rDNA sequences suggest a possible sister-group relationship between the Ulvophyceae and Chlorophyceae, with the Trebouxiophyceae occupying a basal position. On the other hand, separate nuclear SSU rDNA trees (Bhattacharya and Medlin 1998) favor the hypothesis that the Chlorophyceae appeared before the divergence of the Ulvophyceae and Trebouxiophyceae, whereas recent trees, including a wider diversity of ulvophytes (Friedl and O'Kelly 2002) failed to revolve the branching order of the Trebouxiophyceae, Chlorophyceae, and Ulvophyceae. Moreover, other nuclear SSU rDNA trees, including several ulvophytes (Watanabe, Kuroda, and Maiwa 2001) are in agreement with an earlier report (Zechman et al. 1990) and with the concept of the Ulvophyceae sensu Sluiman (1989) in supporting the notion that the ulvophytes are nonmonophyletic. In the Ulvophyceae sensu Sluiman, ulvophytes and trebouxiophytes are merged to form a green-algal group with a counterclockwise arrangement of kinetid components. The analyses supporting the monophyly of ulvophytes suffer from a relatively poor and/or biased taxon sampling (all five orders recognized in this class were not represented), whereas those supporting their nonmonophyly may be plagued with long-branch attraction artifacts.
In this study, we report the complete mtDNA sequence of Pseudendoclonium akinetum, a unicellular member of the Ulvophyceae that belongs to a putatively deep-branching lineage (Floyd and O'Kelly 1990). At 95,880 bp, this ulvophyte mtDNA is the largest green-algal mtDNA analyzed thus far. Our phylogenetic analyses provide support for a sister-group relationship between the Ulvophyceae and Chlorophyceae.
Materials and Methods
Strain and Culture Conditions
Pseudendoclonium akinetum (Tupa 1974) was obtained from the University of Texas Algal Culture Collection (UTEX 1912) and grown in modified Volvox medium (McCracken, Nadakavukaren, and Cain 1980) under 12 h light/dark cycles.
Isolation and Sequencing of mtDNA
A+T-rich organellar DNA was separated from nuclear DNA by CsCl-bisbenzimide isopycnic centrifugation (Turmel et al. 1999). After nebulization of this A+T-rich fraction, a plasmid library of DNA fragments (1200 to 2500 bp) was prepared (Lemieux, Otis, and Turmel 2000). Plasmid DNA templates and PCR fragments spanning uncloned regions of Pseudendoclonium mtDNA were sequenced using ABI Prism 373XL and 377 (Applied Biosystems, Foster City, Calif.) DNA sequencers and the ABI Prism Big Dye terminator sequencing kit (Applied Biosystems) as described previously (Lemieux, Otis, and Turmel 2000). Templates that yielded poor sequences under these conditions were subjected to sequencing using the DYEnamic ET (Amersham Pharmacia Biotech, Baie d'Urfé, Canada) and/or the ABI Prism dGTP Big Dye (Applied Biosystems) dye terminator sequencing kits. Sequences were edited and assembled with AUTOASSEMBLER version 2.1.1 (Applied Biosystems).
Genome Analyses
Gene content was determined by Blast homology searches (Altschul et al. 1990) against the nonredundant database of NCBI. Protein-coding genes and open reading frames (ORFs) were localized precisely using ORFFINDER at NCBI and various programs of the GCG Wisconsin version 10.2 package (Accelrys, Burlington, Mass.), whereas sequences coding for tRNAs were identified with tRNAscan-SE 1.23 (Lowe and Eddy 1997). Patterns of codon usage for protein-coding genes and ORFs were compared using CORRESPOND and CODONPREFERENCE from the Wisconsin package and CAI from the EMBOSS version 2.6.0 package (http://www.hgmp.mrc.ac.uk/Software/EMBOSS/). Repeated sequence elements were identified with PIPMAKER (Schwartz et al. 2000) and REPUTER version 2.74 (Kurtz et al. 2001) and classified with REPEATFINDER (Volfovsky, Haas, and Salzberg 2001).
Phylogenetic Analyses
Mitochondrial genome sequences were retrieved from GenBank: Pseudendoclonium akinetum (accession number AY359242 [this study]), Mesostigma viride (accession number AF353999), Nephroselmis olivacea (accession number AF110138), Prototheca wickerhamii (accession number NC_001613), Pedinomonas minor (accession number NC_000892), Scenedesmus obliquus (accession number AF204057), Chlamydomonas eugametos (accession number NC_001872), Chlamydomonas reinhardtii (accession number NC_001638), Chlorogonium elongatum (accession numbers Y13643 and Y13644), Chaetosphaeridium globosum (accession number NC_004118), Marchantia polymorpha (accession number NC_001660), Arabidopsis thaliana (accession number NC_001284), Beta vulgaris (accession number NC_002511), Chondrus crispus (accession number NC_001677), and Porphyra purpurea (accession number NC_002007). Deduced amino acid sequences from individual genes were aligned using ClustalW version 1.81 (Thompson, Higgins, and Gibson 1994). Data sets were prepared by concatenating the alignments of individual proteins and removing the ambiguously aligned regions with GBLOCKS version 0.73b (Castresana 2000). Phylogenetic trees were inferred using maximum-likelihood (ML) and ML-distance methods. ML trees were computed with PROTML in MOLPHY version 2.3b3 (Adachi and Hasegawa 1996) and CODEML in PAML version 3.11 (Yang 1997) using the amino acid substitution models JTT-F, mtREV24-F, and WAG-F (Whelan and Goldman 2001). -distributed rates of substitutions across sites (eight categories) and/or multiple gene (Mgene option) corrections were applied in some CODEML analyses. Local bootstrap probabilities were estimated by resampling of the estimated log-likelihood (RELL) (Adachi and Hasegawa 1996). Confidence assessments (P-values) of tree selections were evaluated by the Approximately Unbiased, Kishino-Hasegawa, and Shimodaira-Hasegawa tests as implemented in CONSEL version 0.1d (Shimodaira and Hasegawa 2001). The effect of invariable sites on topologies was determined by analysis of a trimmed data set of 1,555 positions containing only the variable sites. -corrected ML distances were calculated with TREE-PUZZLE version 5.0.2 (Strimmer and von Haeseler 1996) under the WAG-F model, and distance trees were computed by weighted neighbor-joining as implemented in WEIGHBOR version 1.2 (Bruno, Socci, and Halpern 2000). Support for ML-distance trees was obtained by bootstrapping (100 replications) with PUZZLEBOOT version 1.03 (A. Roger and M. Holder, http://www.tree-puzzle.de).
Comparisons of Amino Acid Substitution Rates in Different Lineages
We used the data set of 2,107 amino acid positions that was employed for the ML and ML-distance analyses. Differences in the rates of amino acid substitutions among lineages were assessed using the binomial test of Gu and Li (1992).
Results
General Features
Pseudendoclonium mtDNA is a circular DNA molecule of 95,880 bp (fig. 1). Its 57 conserved genes and 36 free-standing ORFs (> 60 codons) represent 47.4% and 11.3% of the genome sequence, respectively. A total of seven introns interrupt four genes (atp1, cob, cox1, and rnl). Intergenic sequences range from 5 to 2,663 bp in size, with an average of about 600 bp, and feature similar proportions of A+T (61.4%) as compared with coding regions (60.2%).
FIG. 1. Gene map of Pseudendoclonium mtDNA. Genes on the outside of the map are transcribed in a clockwise direction; those on the inside of the map are transcribed counterclockwise. tRNA genes are indicated by the one-letter amino acid code followed by the anticodon in parentheses (Me, elongator methionine; Mf, initiator methionine). Seven group I introns (open boxes) interrupt conserved genes; six of these introns contain an internal ORF (gray boxes). Only the free-standing ORFs larger than 60 codons are shown
In terms of coding capacity, the mitochondrial genome of Pseudendoclonium closely resembles those of Nephroselmis and Prototheca. Pseudendoclonium mtDNA lacks six of the protein-coding genes encoded by its Nephroselmis counterpart and only three of the protein-coding genes encoded by Prototheca mtDNA (table 1). It encodes two rRNAs, 25 tRNAs, 12 ribosomal proteins, 17 ATP synthase and respiratory chain components, and also a protein involved in the Sec-independent translocation pathway (MttB). The encoded tRNAs each feature a conventional cloverleaf secondary structure and together are sufficient to translate all of the codons in Pseudendoclonium mtDNA using the standard genetic code (table 2). In contrast to Nephroselmis and Prototheca mtDNAs, no gene for 5S rRNA (rrn5) is present in Pseudendoclonium mtDNA.
Table 1 Distribution of Conserved Genes Coding for Proteins and rRNAs in the mtDNAs of Pseudendoclonium and Selected Chlorophytes.
Table 2 Codon Usage in the 30 Conserved Protein-Coding Genes of Pseudendoclonium mtDNA.
Six intron ORFs in Pseudendoclonium mtDNA encode proteins that are homologous to endonucleases/maturases of the LAGLIDADG family (table 3); each of these intron-encoded proteins contains two copies of the LAGLIDADG motif. Two of the 36 free-standing ORFs, orf289 and orf307, also code for putative LAGLIDADG endonucleases/maturases with two copies of the LAGLIDADG motif. The endonuclease potentially encoded by orf289 shows 48% sequence identity over 282 aligned amino acid positions with the protein specified by orf298 in the fourth intron of cox1 (Pacox1.4), whereas the endonuclease potentially encoded by orf307 has no substantial homology with any of the Pseudendoclonium intron-encoded endonucleases/maturases. Of the 34 remaining free-standing ORFs, only the four harboring more than 300 codons, as well as orf61 and orf90, feature a pattern of codon usage that is similar to that of conserved genes (table 2). This observation suggests that these few ORFs may be expressed at the protein level; however, we have not been able to detect any similarity with known gene sequences.
Table 3 Main Features of Pseudendoclonium Mitochondrial Introns.
Pseudendoclonium mtDNA shares only two small gene clusters (rps12-rps10 and trnE(uuc)-trnW(cca) with Nephroselmis mtDNA) and a single cluster (atp1-nad1) with Prototheca mtDNA. The large cluster of ribosomal protein genes found in Nephroselmis mtDNA has been segmented into several clusters in Pseudendoclonium mtDNA, and as observed for the Nephroselmis and Prototheca mtDNAs, the genes for the SSU and LSU rRNAs have retained their continuous structure.
Introns
All seven introns in Pseudendoclonium mtDNA belong to the group I family (table 3). All of these introns, with the exception of Paatp1.1, show similarities with group I introns inserted at equivalent positions in other green-plant and/or fungal mtDNAs (table 4). The latter green-plant mtDNAs include those of the streptophyte Marchantia and of various chlorophytes exhibiting an "ancestral" or a "reduced-derived" pattern of mtDNA evolution. Sequence conservation between the core structures of four Pseudendoclonium introns (Pacox1.1, Pacox1.2, Pacox1.4, and Parnl.1) and their green-algal homologs is substantial (fig. 2).
Table 4 Group I Introns at Identical Gene Locations in Pseudendoclonium and Other mtDNAs.
FIG. 2. Structural similarities between group I introns in Pseudendoclonium mtDNA and their closest homologs in other green algal mtDNAs. Pacox1.1 and Pacox1.2 were compared with the homologous introns in Prototheca mtDNA, Pacox1.4 was aligned with the homologous intron in Mesostigma mtDNA, and Parnl.1 was compared with the homologous intron in Nephroselmis mtDNA. Introns were modeled according to the nomenclature proposed by Burke et al. (1987). Splice sites between exon and intron residues are denoted by arrows. Identical residues found at the same positions in the compared introns are shown in uppercase characters, whereas positions displaying different nucleotides are denoted by dots. Conserved base-pairings are denoted by dashes. The numbers inside the variable loops indicate the sizes of these loops in the compared introns, with the upper number referring to the Pseudendoclonium sequence
Homologs of the Parnl.1 intron have also been identified in the chloroplast rrl genes of several green algae (Turmel et al. 1993). One of these chloroplast introns (Chrnl.1) contains an ORF in L9.1 (as in Parnl.1) that codes for the DNA-homing endonuclease I-ChuI (C?té et al. 1993). The Chrnl.1 and Parnl.1 introns share 69% sequence identity over 169 aligned nucleotides in their core structures, and their encoded proteins show 65.3% amino acid similarities over 219 aligned amino acid positions. This important level of sequence conservation is consistent with our previous finding of closely related group I introns in the chloroplast and mitochondrial genomes of green algae (Turmel et al. 1995; Turmel et al. 1999; Lucas et al. 2001; Turmel, Otis, and Lemieux 2002a) and further supports the idea that lateral transfers of introns have occurred between different organellar compartments during the evolution of green plants.
Repeated Sequence Elements
A comparison of the Pseudendoclonium mtDNA sequence against itself using PIPMAKER (Schwartz et al. 2000) revealed the presence of many degenerated repeated elements within intergenic regions and introns (fig. 3). We identified these repeated sequences using the program REPUTER and found that they are up to 350 nt in size and do not differ substantially from the rest of the Pseudendoclonium genome in terms of nucleotide composition. The repeats that were 50 nt or more were classified using REPEATFINDER (Volfovsky, Haas, and Salzberg 2001); they represent about 10 kb of the genome and form four distinct classes, designated C1 to C4 (fig. 3 and table 5). The members of a given class share no sequence similarity with those of other classes. The first class of repeats (C1) is the largest. Its members map to at least 16 different loci (fig. 3) and can be further classified into 14 subclasses, designated C1R1 through C1R14. The longest repeat within each subclass, defined as the prototype, is made up of repeated units (subrepeats) that also constitute the shorter repeated elements found in the same subclass. Distinct subclasses feature different arrangements of repeated units. The coordinates of the prototypes and number of repeated elements within each subclass are given in table 5. The abundance of subclasses in class 1 suggests that numerous recombination and shuffling events between repeated units took place during the evolution of the mitochondrial genome. On the other hand, classes 2, 3, and 4 are much less complex than class 1. Each exhibits only one subclass of repeats, with two identical copies of the prototype sequence that are far apart from one another. It is likely that these elements arose from single duplication events. In addition to the repeats described above, Pseudendoclonium mtDNA features numerous repeated elements less than 50 nt that span about 4 kb of this genome (fig. 3).
FIG. 3. Positions of repeated sequence elements in Pseudendoclonium mtDNA as revealed by PIPMAKER. Repeats of 50 nt or more were sorted into four classes by REPEATFINDER. Repeats less than 50 nt were not classified. Genes and their polarities are denoted by horizontal arrows, and exons are represented by filled boxes. Similarities between repeated sequence elements are shown as average percentage identity (between 50% to 100% identity)
Table 5 Prototypes of Repeated Sequence Elements of 50 nt or More in Pseudendoclonium mtDNA.
Phylogenetic Analyses
The amino acid sequences derived from the seven protein-coding genes (cob, cox1, nad1, nad2, nad4, nad5, and nad6) that are common to the mtDNAs of Pseudendoclonium and 11 other green plants were concatenated and analyzed with ML and ML-distance methods of phylogenetic inference, using as outgroup the homologous sequences from Mesostigma. Note that when the red algae Porphyra purpurea and Chondrus crispus were used as outgroup, we observed no change in the position of Pseudendoclonium. The WAG-F model of amino acid replacement was selected for all phylogenetic analyses, because it gave higher likelihood values than the JTT-F and mtREV24-F models (table 6). The ML and ML-distance trees inferred by assuming -distributed rates of substitutions across sites revealed a strong affiliation of Pseudendoclonium with the highly supported clade containing Pedinomonas and the four chlorophycean green algae whose mtDNA sequences has been determined to date (fig. 4). As shown by their very long branches, the members of this clade, designated here "reduced-derived" clade, display a higher rate of mtDNA sequence evolution compared to the other green plants examined. Phylogenetic relationships among members of the "reduced-derived" clade were found to be well resolved in all analyses, and the affiliation of Pseudendoclonium with this clade remained strongly supported when we excluded invariable sites and/or when we included an additional parameter (Mgene option in CODEML) to take into account the different evolutionary rates of the selected mitochondrial proteins.
Table 6 Tree-Related Statistics and Log-Likelihood Values for the Mitochondrial Data Set of 2,107 Amino Acid Positions.
FIG. 4. Phylogenetic position of Pseudendoclonium as inferred from the deduced amino acid sequences of seven mitochondrial genes. ML and ML-distances analyses of the data set (2,107 amino acid positions) were carried out under the WAG-F model of amino acid replacement, assuming -distributed rates of substitutions across sites. The best-supported ML tree computed with CODEML is shown. Numbers above the nodes indicate support values; upper and lower values are from the ML and ML-distance analyses, respectively. The chlorophyte lineages displaying a "reduced-derived" pattern of mtDNA evolution are represented as thick lines
To our surprise, we found that under certain conditions of phylogenetic analyses the "reduced-derived" clade groups with the rest of green algae and land plants. For instance, in the best ML tree shown in figure 4, the cluster formed by Pseudendoclonium and the "reduced-derived" clade occupies the Chlorophyta lineage. This result contrasts with previously reported phylogenies in which members of the "reduced-derived" clade branch consistently outside the monophyletic group formed by the rest of green plants, most certainly as a result of long-branch attraction artifacts (Turmel, Otis, and Lemieux 1999; Nedelcu et al. 2000). We explored the phylogenetic conditions that contribute to the recovery of the correct branching pattern in our best ML tree and found that the selected inference method, evolutionary model, and taxon sampling all play an important role in the analyses. Analyses based on ML-distances and performed under the JTT-F and mtREV24-F models failed to recover the correct topology as the best tree. Moreover, the inclusion of the Pseudendoclonium taxon in the concatenated data set and the addition of a correction for amino acid substitutions across sites also represent important factors in the ML analyses. However, even under the most favorable ML conditions for recovering the correct tree, alternate topologies in which the "reduced-derived" cluster branches outside the clade formed by the rest of green plants were detected relatively frequently among the RELL bootstrap replicates. These alternate topologies proved to be not significantly different (P < 0.1) from the best tree in the Kishino-Hasegawa and the Shimodaira-Hasegawa tests.
Rates of Amino Acid Substitutions
Considering the branch lengths of the best ML tree (fig. 4), it appears that Pseudendoclonium mtDNA evolves at a slower rate than the mtDNAs of taxa from the "reduced-derived" clade. To determine whether this interpretation is correct, we assessed the relative rates of amino acid substitutions of mtDNA-encoded proteins among the different lineages using the binomial test of Gu and Li (1992) (table 7). The substitution rates of Pseudendoclonium proteins were found to be significantly smaller (P < 0.001) than those observed for Scenedesmus and other green algae from the "reduced-derived" clade. Pseudendoclonium mtDNA–encoded proteins, however, evolve significantly faster (P < 0.001) than their Nephroselmis and Prototheca counterparts.
Table 7 Differences in the Number of Amino Acid Substitutions in Seven Mitochondrial Proteins and Test for Equal Rates of Substitutions in Selected Pairs of Green Algal Lineages.
Discussion
Distinctive Features of Pseudendoclonium mtDNA
Of all the green-algal mtDNAs sequenced to date, Pseudendoclonium mtDNA is the genome with the largest size and the lowest gene density (table 8). At 95,880 bp, the size of this genome is almost twofold larger than those of the two chlorophytes displaying an ancestral pattern of evolution (Nephroselmis and Prototheca). Despite this substantial expansion in size, Pseudendoclonium mtDNA displays a smaller repertoire of conserved genes compared with its Nephroselmis and Prototheca counterparts (table 8). Most of its increased size is accounted for by dispersed repeats and sequences of unknown nature/origin that reside mainly within the intergenic regions.
Table 8 Compared Features of Pseudendoclonium mtDNA and Other Green-Plant mtDNAs.
The unusually large size and low gene density of Pseudendoclonium mtDNA, together with the presence of dispersed repeats, is reminiscent of the "expanded" pattern of evolution exhibited by embryophyte mtDNAs. Both the expanded Pseudendoclonium and the embryophyte mtDNAs encode a similar number of conserved genes, although they differ in their gene repertoire; moreover, most of their extraneous DNA sequences are of unknown origin and/or function (Unseld et al. 1997; Kubo et al. 2000; Notsu et al. 2002). The finding that Pseudendoclonium mtDNA is much larger than its chlorophyte counterparts suggests that mitochondrial genome expansion occurred independently in the Chlorophyta and Streptophyta. Analyses of additional ulvophyte mtDNAs will be required to determine whether the Ulvophyceae displays the pattern of progressive genome expansion observed in the Streptophyta.
On one hand, the gene content (a relatively complex gene repertoire) and gene structure (lack of fragmented and scrambled rRNA genes) of Pseudendoclonium mtDNA are characteristic of "ancestral" mtDNAs. On the other hand, its low gene density, abundant repeats, and the absence of certain genes (nad9, rpl6, rps7, and rrn5) are typical of Scenedesmus and Pedinomonas mtDNAs. The finding of genomic features that are typical of the "reduced-derived" pattern of mtDNA evolution, together with the presence of ancestral features, suggests that Pseudendoclonium belongs to a lineage that appeared after the emergence of the Trebouxiophyceae but before the divergence of the Chlorophyceae. Further supporting this notion are three independent observations. First, ML and ML-distance trees inferred from mtDNA-encoded proteins always cluster Pseudendoclonium with chlorophycean green algae and consistently place the trebouxiophyte Prototheca at the base of this clade (see next section). Second, the overall rate of sequence evolution appears to be accelerated to an intermediary level in Pseudendoclonium mtDNA as compared with the slow rates observed in "ancestral" mtDNAs and the very fast rates detected in Pedinomonas, Scenedesmus and chlorophycean green-algal mtDNAs (fig. 4). Third, close relatives of the Pacox1.2, Pacox1.4, and Parnl.1 introns are present in mtDNAs displaying both the "ancestral" and "reduced-derived" patterns of evolution.
Phylogenetic Niche of the Ulvophyceae
Our phylogenetic analyses of concatenated mtDNA-encoded protein sequences reveal a close relationship between Pseudendoclonium and chlorophycean green algae, with the trebouxiophyte Prototheca occupying a basal position (fig. 4). These analyses included all the green-algal mtDNA sequences available in public databases to minimize possible undesirable effects of small taxon sampling and used Mesostigma viride as outgroup to maximize the amount of phylogenetic information. The position of Pseudendoclonium relative to the Chlorophyceae and Trebouxiophyceae may be the result of genuine phylogenetic signal because it is consistent with our finding that Pseudendoclonium mtDNA shares derived structural features with its homologs in chlorophycean green algae. However, the branching order of the Chlorophyceae/Ulvophyceae/Trebouxiophyceae remains an unresolved issue. The tree recovered in our analyses may reflect variations in the evolutionary rates of mtDNA sequences rather than the underlying phylogenetic signal, and although we have included all currently available green-algal mtDNAs in our analyses, taxon sampling is still very low, with only one representative of the Ulvophyceae and of the Trebouxiophyceae.
Pseudendoclonium appears to be closely related to Pedinomonas, a green alga of uncertain affiliation. In mitochondrial trees, Pedinomonas branches immediately after the emergence of the Pseudendoclonium lineage. Given the very long branch displayed by Pedinomonas, the phylogenetic position of this chlorophyte might be attributed to long-branch artifacts. However, the existence of a close relationship between Pedinomonas and ulvophytes is supported by the observation that the basal bodies of Pedinomonas and ulvophytes display an absolute counterclockwise orientation that contrasts with the directly opposed or clockwise orientation found in chlorophycean green algae (Melkonian 1990). Considering that the 25-kb mtDNA of Pedinomonas is highly reduced in size and gene content relative to its Pseudendoclonium and Scenedesmus relatives and also considering the much longer branch displayed by Pedinomonas mtDNA, it appears that the mitochondrial genome evolved at a more accelerated rate in the lineage leading to Pedinomonas than in the Pseudendoclonium and Scenedesmus lineages.
Repeated Sequence Elements As an Evolutionary Force in the Chlorophyta
Small repeats have been previously identified in chlorophyte mtDNAs. The few that are found in Prototheca mtDNA vary from 30 to 200 nt, are mostly arranged in tandem, and the recurring motifs are rich in A+T (Wolff et al. 1994). Repeats in Scenedesmus mtDNA range from 16 to 118 nt, and, like their Pseudendoclonium counterparts, account for at least 15% of the genome, flank many individual genes, are composed of various subrepeats, and show a low bias in base composition (Nedelcu et al. 2000). The repeats harbored by chlamydomonad mtDNAs are shorter (9 to 14 nt) and richer in G+C (Boer and Gray 1991; Nedelcu and Lee 1998). Unlike all their known chlorophyte counterparts, the numerous repeated sequences found in Pedinomonas mtDNA are densely packed within a discrete, noncoding region of the genome (Turmel et al. 1999). The low abundance of repeats in Nephroselmis and Prototheca mtDNAs, together with the increased occurrence of these elements in the more derived lineages leading to Pseudendoclonium and Scenedesmus, raise the question on their origin. Repeats might have been present in the mitochondrial genome of the last common ancestor of the ulvophytes and chlorophycean green algae, and have diverged after the split of these lineages. Alternatively, they might have arisen independently in the Ulvophyceae and Chlorophyceae.
Considering that recombination between dispersed repeats can lead to genome rearrangements (gene losses or inversions) and that gene content became reduced as families of dispersed repeats emerged and grew in size during the evolution of chlorophyte mtDNAs (at least in derived lineages), we speculate that repeated elements have played an important role in generating the great diversity of size and gene arrangement seen in these mtDNAs. In chlamydomonad mtDNAs, where gene content is the poorest of the Chlorophyta lineage, excision of coding regions via recombination between flanking short repeats has been invoked to explain their reduced gene content (Nedelcu 1997; Nedelcu 1998). Dispersed repeats are also thought to act as hot spots for recombination in land-plant (Palmer and Herbon 1988; Mackenzie, He, and Lyznik 1994), fungal (Jamet-Vierny, Boulay, and Briand 1997), and animal (Lunt and Hyman 1997) mtDNAs. Aside from dispersed repeats, other factors most probably determine the tempo of gene loss. For angiosperms, it has been shown that the tempo of mitochondrial gene loss (and probably gene transfer to the nucleus) is remarkably punctuated. Certain lineages have rapidly lost most or all of their 16 ribosomal protein and sdh genes, whereas other lineages, mostly ancient lineages, have maintained a constant set of mitochondrial genes for hundreds of millions of years (Adams et al. 2002). This punctuated pattern appears to be driven by major episodic rises in the rate of functional gene transfer.
Interestingly, short, dispersed repeats have been observed in the chloroplast DNAs of Chlamydomonas taxa (Boudreau and Turmel 1996; Maul et al. 2002) and of the trebouxiophyte Chlorella vulgaris (Maul et al. 2002) but not in Nephroselmis chloroplast DNA (Maul et al. 2002). The phylogenetic distribution of small, repeated elements within chlorophyte chloroplast DNAs thus parallels that observed for the mitochondrial genome. Given the evidence for lateral transfers of introns (genetic elements often associated with short repeats) between the mitochondrial and chloroplast compartments (Turmel et al. 1995; Turmel et al. 1999; Lucas et al. 2001; Turmel, Otis, and Lemieux 2002a), it is possible that such events contribute to the dispersal of short repeats and account for the presence of these elements in both organelle genomes of the same cell. In this context, it will be interesting to see if short, repeated elements are found in ulvophyte chloroplast DNAs.
Conclusion
Representing the first ulvophyte organelle genome sequence publicly available, Pseudendoclonium mtDNA is a valuable source of information to our understanding of mitochondrial genome evolution in the Chlorophyta. It will be important to examine mtDNAs from additional representatives of the Ulvophyceae to determine if the distinctive traits exhibited by Pseudendoclonium mtDNA are conserved in other ulvophyte mtDNAs or are restricted to the lineage leading to this ulvophyte. A broader study of mitochondrial genomes, including more trebouxiophytes and chlorophycean green algae, will be required to resolve the question about the monophyly of the Ulvophyceae, to clarify the interrelationships between the Chlorophyceae, Trebouxiophyceae, and Ulvophyceae, and also to better understand how the great diversity seen at the mitochondrial genome level arose during the evolution of chlorophytes.
Supplementary Material
The genome sequence reported in this paper has been deposited in the GenBank database (accession number AY359242).
Acknowledgements
This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (to M.T. and C.L.). J.-F.P. gratefully acknowledges a scholarship from CREFSIP (Centre de Recherche sur la Fonction, la Structure et l'Ingénierie des Protéines).
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E-mail: Monique.Turmel@rsvs.ulaval.ca.
Abstract
The mitochondrial genome has undergone radical changes in both the Chlorophyta and Streptophyta, yet little is known about the dynamics of mtDNA evolution in either of these lineages. In the Chlorophyta, which comprises four of the five recognized classes of green algae (Prasinophyceae, Trebouxiophyceae, Ulvophyceae, and Chlorophyceae), the mitochondrial genome varies from 16 to 55 kb. This genome has retained a compact gene organization and a relatively complex gene repertoire ("ancestral" pattern) in the basal lineages represented by the Trebouxiophyceae and Prasinophyceae, whereas it has been reduced in size and gene complement and tends to evolve much more rapidly at the sequence level ("reduced-derived" pattern of evolution) in the Chlorophyceae and the lineage leading to the enigmatic chlorophyte Pedinomonas. To gain information about the evolutionary trends of mtDNA in the Ulvophyceae and also to gain insights into the phylogenetic relationships between ulvophytes and other chlorophytes, we have determined the mtDNA sequence of Pseudendoclonium akinetum. At 95,880 bp, Pseudendoclonium mtDNA is the largest green-algal mitochondrial genome sequenced to date and has the lowest gene density. These derived features are reminiscent of the "expanded" pattern exhibited by embryophyte mtDNAs, indicating that convergent evolution towards genome expansion has occurred independently in the Chlorophyta and Streptophyta. With 57 conserved genes, the gene repertoire of Pseudendoclonium mtDNA is slightly smaller than those of the prasinophyte Nephroselmis olivacea and the trebouxiophyte Prototheca wickerhamii. This ulvophyte mtDNA contains seven group I introns, four of which have homologs in green-algal mtDNAs displaying an "ancestral" or a "reduced-derived" pattern of evolution. Like its counterpart in the chlorophycean green alga Scenedesmus obliquus, it features numerous small, dispersed repeats in intergenic regions and introns. Its overall rate of sequence evolution appears to be accelerated to an intermediary level as compared with the rates observed in "ancestral" and "reduced-derived" mtDNAs. In agreement with the finding that Pseudendoclonium mtDNA exhibits features typical of both the "ancestral" and "reduced-derived" patterns of evolution, phylogenetic analyses of seven mtDNA-encoded proteins revealed a sister-group relationship between this ulvophyte and chlorophytes displaying "reduced-derived" mtDNAs.
Key Words: Green algae ? Ulvophyceae ? Pseudendoclonium akinetum ? mitochondrial DNA ? group I introns ? repeated sequences
Introduction
The mitochondrial genomes of green plants are highly variable in size, gene content, and organization and show divergent evolutionary trends in some lineages, as revealed by the complete mtDNA sequences that have been reported thus far for 13 members of this group. Representatives of the two main phyla of green plants, the Streptophyta and Chlorophyta, have been examined in these genome analyses. The Streptophyta (Bremer 1985) comprises all embryophytes (land plants) and their closest green-algal relatives, the members of the class Charophyceae sensu Mattox and Stewart (1984). In this phylum, the mitochondrial genomes of the bryophyte Marchantia polymorpha (Oda et al. 1992), of three angiosperms (Arabidopsis thaliana [Unseld et al. 1997], Beta vulgaris [Kubo et al. 2000], and Oryza sativa [Notsu et al. 2002]), and of the charophycean green alga Chaetosphaeridium globosum (Turmel, Otis, and Lemieux 2002b) have been entirely sequenced. The Chlorophyta (Sluiman 1985) comprises the four other classes of green algae: the Prasinophyceae, Ulvophyceae, Trebouxiophyceae, and Chlorophyceae. The seven chlorophyte mitochondrial DNA (mtDNA) sequences that are currently available include those of four chlorophycean green algae (Chlamydomonas reinhardtii [Michaelis, Vahrenholz, and Pratje 1990], Chlamydomonas eugametos [Denovan-Wright, Nedelcu, and Lee 1998], Chlorogonium elongatum [Kroymann and Zetsche 1998], and Scenedesmus obliquus [Kück, Jekosch, and Holzamer 2000; Nedelcu et al. 2000]), the nonphotosynthetic trebouxiophyte Prototheca wickerhamii (Wolff et al. 1994), the prasinophyte Nephroselmis olivacea (Turmel et al. 1999), and a chlorophyte of uncertain phylogenetic affinity, Pedinomonas minor (Turmel et al. 1999). The remaining green alga whose mitochondrial genome has been scrutinized, Mesostigma viride (Turmel, Otis, and Lemieux 2002a), belongs to the Streptophyta (Bhattacharya et al. 1998; Marin and Melkonian 1999; Karol et al. 2001) or to a lineage that emerged before the divergence of the Streptophyta and Chlorophyta (Lemieux, Otis, and Turmel 2000; Turmel et al. 2002; Turmel, Otis, and Lemieux 2002a).
Because of their large size and their tendency to incorporate foreign DNA (from the nucleus and the chloroplast), land-plant mitochondrial genomes have been reported to feature an "expanded" pattern of evolution (Turmel et al. 1999). These mitochondrial genomes are the largest (187 kb in Marchantia to 2,000 kb in muskmelon) among green plants, and they also show the greatest structural complexity. Most of the increased size of land-plant mtDNAs relative to their green-algal homologs is accounted for by noncoding sequences that reside either in intergenic regions or introns (Oda et al. 1992; Unseld et al. 1997; Kubo et al. 2000; Notsu et al. 2002). Sixty-nine mitochondrial genes have been identified in Marchantia (Oda et al. 1992; Gray et al. 1998), whereas about 50 have been found in angiosperms (Unseld et al. 1997; Kubo et al. 2000; Notsu et al. 2002). This substantial difference in coding capacity is attributed to gene transfer to the nucleus, a widespread and ongoing phenomenon (Schuster and Brennicke 1994; Palmer et al. 2000; Adams et al. 2002). In embryophyte mitochondria, unicircular genome-sized molecules coexist in a dynamic equilibrium with subgenomic circles (Palmer and Shields 1984; Mackenzie, He, and Lyznik 1994). In Marchantia mitochondria, unicircular-genome sized molecules apparently coexist with linear molecules and complex branched structures of multigenomic sizes (Oldenburg and Bendich 2001). In contrast to their fluid structure, land-plant mitochondrial genomes evolve extremely slowly at the sequence level; in angiosperm mitochondria, point mutations can occur at a frequency up to 100 times lower than in vertebrate mitochondria (Wolfe, Li, and Sharp 1987; Palmer and Herbon 1988).
The more compact green-algal mitochondrial genomes display distinctive evolutionary patterns. They range in size from 16 kb (in C. reinhardtii) to 67.8 kb (in Mesostigma) and encode 12 (in C. reinhardtii, C. eugametos, and Chlorogonium) to 67 (in Chaetosphaeridium) genes. An "ancestral" (minimally derived) evolutionary pattern (Turmel et al. 1999) has been ascribed to the circular-mapping mtDNAs of Mesostigma, Chaetosphaeridium, Nephroselmis, and Prototheca, because of their large number of conserved genes (>60), their high gene density, and their important sequence conservation. Not only fewer genes but also a greater variability in gene content (12 to 42 genes) and structural organization (linear or circular-mapping molecules, or even multimeric molecules, as reported for Polytomella [Fan and Lee 2002]) have been found in chlorophycean green-algal mtDNAs. The coding sequences of these genomes are highly divergent from those of other green plants and feature numerous deletions/additions; moreover, the rRNA gene-coding regions are fragmented into pieces that are dispersed throughout the genomes. As a consequence of this high sequence divergence, chlorophycean taxa exhibit very long branches in mitochondrial trees, and, most probably because of long-branch attraction artifacts, usually lie outside the green-plant clade when other green plants and non–green-plant taxa are included in the analyses. A "reduced-derived pattern" (Turmel et al. 1999) of evolution has been assigned to the three smallest and gene-poorest chlorophycean mtDNAs (i.e., to C. reinhardtii, C. eugametos, and Chlorogonium mtDNAs). Because of its less-derived characters, the Scenedesmus mtDNA sequence has been considered to display an "intermediate" pattern of evolution (Nedelcu et al. 2000).
The present study was undertaken to gather information about the evolutionary trends of the mitochondrial genome in the Ulvophyceae and also to gain insights into the phylogenetic relationships between ulvophytes and other chlorophytes. Various hypotheses have been proposed concerning the phylogenetic position of the Ulvophyceae within the Chlorophyta, but none is strongly supported by statistical analyses. On the basis of ultrastructural studies (Mattox and Stewart 1984; O'Kelly and Floyd 1984) and some phylogenetic analyses of nuclear small subunit (SSU) rDNA sequences (Friedl 1995; Bhattacharya, Friedl, and Damberger 1996; Nakayama, Watanabe, and Inouye 1996; Chapman et al. 1998; Watanabe et al. 2000; Wolf et al. 2002), it has been proposed that the Ulvophyceae are a monophyletic assemblage that emerged before the divergence of the Trebouxiophyceae and Chlorophyceae. Independent inferences from nuclear SSU rDNA sequences (Friedl 1997; Marin and Melkonian 1999) and from concatenated chloroplast SSU and large subunit (LSU) (Turmel et al. 2002) rDNA sequences suggest a possible sister-group relationship between the Ulvophyceae and Chlorophyceae, with the Trebouxiophyceae occupying a basal position. On the other hand, separate nuclear SSU rDNA trees (Bhattacharya and Medlin 1998) favor the hypothesis that the Chlorophyceae appeared before the divergence of the Ulvophyceae and Trebouxiophyceae, whereas recent trees, including a wider diversity of ulvophytes (Friedl and O'Kelly 2002) failed to revolve the branching order of the Trebouxiophyceae, Chlorophyceae, and Ulvophyceae. Moreover, other nuclear SSU rDNA trees, including several ulvophytes (Watanabe, Kuroda, and Maiwa 2001) are in agreement with an earlier report (Zechman et al. 1990) and with the concept of the Ulvophyceae sensu Sluiman (1989) in supporting the notion that the ulvophytes are nonmonophyletic. In the Ulvophyceae sensu Sluiman, ulvophytes and trebouxiophytes are merged to form a green-algal group with a counterclockwise arrangement of kinetid components. The analyses supporting the monophyly of ulvophytes suffer from a relatively poor and/or biased taxon sampling (all five orders recognized in this class were not represented), whereas those supporting their nonmonophyly may be plagued with long-branch attraction artifacts.
In this study, we report the complete mtDNA sequence of Pseudendoclonium akinetum, a unicellular member of the Ulvophyceae that belongs to a putatively deep-branching lineage (Floyd and O'Kelly 1990). At 95,880 bp, this ulvophyte mtDNA is the largest green-algal mtDNA analyzed thus far. Our phylogenetic analyses provide support for a sister-group relationship between the Ulvophyceae and Chlorophyceae.
Materials and Methods
Strain and Culture Conditions
Pseudendoclonium akinetum (Tupa 1974) was obtained from the University of Texas Algal Culture Collection (UTEX 1912) and grown in modified Volvox medium (McCracken, Nadakavukaren, and Cain 1980) under 12 h light/dark cycles.
Isolation and Sequencing of mtDNA
A+T-rich organellar DNA was separated from nuclear DNA by CsCl-bisbenzimide isopycnic centrifugation (Turmel et al. 1999). After nebulization of this A+T-rich fraction, a plasmid library of DNA fragments (1200 to 2500 bp) was prepared (Lemieux, Otis, and Turmel 2000). Plasmid DNA templates and PCR fragments spanning uncloned regions of Pseudendoclonium mtDNA were sequenced using ABI Prism 373XL and 377 (Applied Biosystems, Foster City, Calif.) DNA sequencers and the ABI Prism Big Dye terminator sequencing kit (Applied Biosystems) as described previously (Lemieux, Otis, and Turmel 2000). Templates that yielded poor sequences under these conditions were subjected to sequencing using the DYEnamic ET (Amersham Pharmacia Biotech, Baie d'Urfé, Canada) and/or the ABI Prism dGTP Big Dye (Applied Biosystems) dye terminator sequencing kits. Sequences were edited and assembled with AUTOASSEMBLER version 2.1.1 (Applied Biosystems).
Genome Analyses
Gene content was determined by Blast homology searches (Altschul et al. 1990) against the nonredundant database of NCBI. Protein-coding genes and open reading frames (ORFs) were localized precisely using ORFFINDER at NCBI and various programs of the GCG Wisconsin version 10.2 package (Accelrys, Burlington, Mass.), whereas sequences coding for tRNAs were identified with tRNAscan-SE 1.23 (Lowe and Eddy 1997). Patterns of codon usage for protein-coding genes and ORFs were compared using CORRESPOND and CODONPREFERENCE from the Wisconsin package and CAI from the EMBOSS version 2.6.0 package (http://www.hgmp.mrc.ac.uk/Software/EMBOSS/). Repeated sequence elements were identified with PIPMAKER (Schwartz et al. 2000) and REPUTER version 2.74 (Kurtz et al. 2001) and classified with REPEATFINDER (Volfovsky, Haas, and Salzberg 2001).
Phylogenetic Analyses
Mitochondrial genome sequences were retrieved from GenBank: Pseudendoclonium akinetum (accession number AY359242 [this study]), Mesostigma viride (accession number AF353999), Nephroselmis olivacea (accession number AF110138), Prototheca wickerhamii (accession number NC_001613), Pedinomonas minor (accession number NC_000892), Scenedesmus obliquus (accession number AF204057), Chlamydomonas eugametos (accession number NC_001872), Chlamydomonas reinhardtii (accession number NC_001638), Chlorogonium elongatum (accession numbers Y13643 and Y13644), Chaetosphaeridium globosum (accession number NC_004118), Marchantia polymorpha (accession number NC_001660), Arabidopsis thaliana (accession number NC_001284), Beta vulgaris (accession number NC_002511), Chondrus crispus (accession number NC_001677), and Porphyra purpurea (accession number NC_002007). Deduced amino acid sequences from individual genes were aligned using ClustalW version 1.81 (Thompson, Higgins, and Gibson 1994). Data sets were prepared by concatenating the alignments of individual proteins and removing the ambiguously aligned regions with GBLOCKS version 0.73b (Castresana 2000). Phylogenetic trees were inferred using maximum-likelihood (ML) and ML-distance methods. ML trees were computed with PROTML in MOLPHY version 2.3b3 (Adachi and Hasegawa 1996) and CODEML in PAML version 3.11 (Yang 1997) using the amino acid substitution models JTT-F, mtREV24-F, and WAG-F (Whelan and Goldman 2001). -distributed rates of substitutions across sites (eight categories) and/or multiple gene (Mgene option) corrections were applied in some CODEML analyses. Local bootstrap probabilities were estimated by resampling of the estimated log-likelihood (RELL) (Adachi and Hasegawa 1996). Confidence assessments (P-values) of tree selections were evaluated by the Approximately Unbiased, Kishino-Hasegawa, and Shimodaira-Hasegawa tests as implemented in CONSEL version 0.1d (Shimodaira and Hasegawa 2001). The effect of invariable sites on topologies was determined by analysis of a trimmed data set of 1,555 positions containing only the variable sites. -corrected ML distances were calculated with TREE-PUZZLE version 5.0.2 (Strimmer and von Haeseler 1996) under the WAG-F model, and distance trees were computed by weighted neighbor-joining as implemented in WEIGHBOR version 1.2 (Bruno, Socci, and Halpern 2000). Support for ML-distance trees was obtained by bootstrapping (100 replications) with PUZZLEBOOT version 1.03 (A. Roger and M. Holder, http://www.tree-puzzle.de).
Comparisons of Amino Acid Substitution Rates in Different Lineages
We used the data set of 2,107 amino acid positions that was employed for the ML and ML-distance analyses. Differences in the rates of amino acid substitutions among lineages were assessed using the binomial test of Gu and Li (1992).
Results
General Features
Pseudendoclonium mtDNA is a circular DNA molecule of 95,880 bp (fig. 1). Its 57 conserved genes and 36 free-standing ORFs (> 60 codons) represent 47.4% and 11.3% of the genome sequence, respectively. A total of seven introns interrupt four genes (atp1, cob, cox1, and rnl). Intergenic sequences range from 5 to 2,663 bp in size, with an average of about 600 bp, and feature similar proportions of A+T (61.4%) as compared with coding regions (60.2%).
FIG. 1. Gene map of Pseudendoclonium mtDNA. Genes on the outside of the map are transcribed in a clockwise direction; those on the inside of the map are transcribed counterclockwise. tRNA genes are indicated by the one-letter amino acid code followed by the anticodon in parentheses (Me, elongator methionine; Mf, initiator methionine). Seven group I introns (open boxes) interrupt conserved genes; six of these introns contain an internal ORF (gray boxes). Only the free-standing ORFs larger than 60 codons are shown
In terms of coding capacity, the mitochondrial genome of Pseudendoclonium closely resembles those of Nephroselmis and Prototheca. Pseudendoclonium mtDNA lacks six of the protein-coding genes encoded by its Nephroselmis counterpart and only three of the protein-coding genes encoded by Prototheca mtDNA (table 1). It encodes two rRNAs, 25 tRNAs, 12 ribosomal proteins, 17 ATP synthase and respiratory chain components, and also a protein involved in the Sec-independent translocation pathway (MttB). The encoded tRNAs each feature a conventional cloverleaf secondary structure and together are sufficient to translate all of the codons in Pseudendoclonium mtDNA using the standard genetic code (table 2). In contrast to Nephroselmis and Prototheca mtDNAs, no gene for 5S rRNA (rrn5) is present in Pseudendoclonium mtDNA.
Table 1 Distribution of Conserved Genes Coding for Proteins and rRNAs in the mtDNAs of Pseudendoclonium and Selected Chlorophytes.
Table 2 Codon Usage in the 30 Conserved Protein-Coding Genes of Pseudendoclonium mtDNA.
Six intron ORFs in Pseudendoclonium mtDNA encode proteins that are homologous to endonucleases/maturases of the LAGLIDADG family (table 3); each of these intron-encoded proteins contains two copies of the LAGLIDADG motif. Two of the 36 free-standing ORFs, orf289 and orf307, also code for putative LAGLIDADG endonucleases/maturases with two copies of the LAGLIDADG motif. The endonuclease potentially encoded by orf289 shows 48% sequence identity over 282 aligned amino acid positions with the protein specified by orf298 in the fourth intron of cox1 (Pacox1.4), whereas the endonuclease potentially encoded by orf307 has no substantial homology with any of the Pseudendoclonium intron-encoded endonucleases/maturases. Of the 34 remaining free-standing ORFs, only the four harboring more than 300 codons, as well as orf61 and orf90, feature a pattern of codon usage that is similar to that of conserved genes (table 2). This observation suggests that these few ORFs may be expressed at the protein level; however, we have not been able to detect any similarity with known gene sequences.
Table 3 Main Features of Pseudendoclonium Mitochondrial Introns.
Pseudendoclonium mtDNA shares only two small gene clusters (rps12-rps10 and trnE(uuc)-trnW(cca) with Nephroselmis mtDNA) and a single cluster (atp1-nad1) with Prototheca mtDNA. The large cluster of ribosomal protein genes found in Nephroselmis mtDNA has been segmented into several clusters in Pseudendoclonium mtDNA, and as observed for the Nephroselmis and Prototheca mtDNAs, the genes for the SSU and LSU rRNAs have retained their continuous structure.
Introns
All seven introns in Pseudendoclonium mtDNA belong to the group I family (table 3). All of these introns, with the exception of Paatp1.1, show similarities with group I introns inserted at equivalent positions in other green-plant and/or fungal mtDNAs (table 4). The latter green-plant mtDNAs include those of the streptophyte Marchantia and of various chlorophytes exhibiting an "ancestral" or a "reduced-derived" pattern of mtDNA evolution. Sequence conservation between the core structures of four Pseudendoclonium introns (Pacox1.1, Pacox1.2, Pacox1.4, and Parnl.1) and their green-algal homologs is substantial (fig. 2).
Table 4 Group I Introns at Identical Gene Locations in Pseudendoclonium and Other mtDNAs.
FIG. 2. Structural similarities between group I introns in Pseudendoclonium mtDNA and their closest homologs in other green algal mtDNAs. Pacox1.1 and Pacox1.2 were compared with the homologous introns in Prototheca mtDNA, Pacox1.4 was aligned with the homologous intron in Mesostigma mtDNA, and Parnl.1 was compared with the homologous intron in Nephroselmis mtDNA. Introns were modeled according to the nomenclature proposed by Burke et al. (1987). Splice sites between exon and intron residues are denoted by arrows. Identical residues found at the same positions in the compared introns are shown in uppercase characters, whereas positions displaying different nucleotides are denoted by dots. Conserved base-pairings are denoted by dashes. The numbers inside the variable loops indicate the sizes of these loops in the compared introns, with the upper number referring to the Pseudendoclonium sequence
Homologs of the Parnl.1 intron have also been identified in the chloroplast rrl genes of several green algae (Turmel et al. 1993). One of these chloroplast introns (Chrnl.1) contains an ORF in L9.1 (as in Parnl.1) that codes for the DNA-homing endonuclease I-ChuI (C?té et al. 1993). The Chrnl.1 and Parnl.1 introns share 69% sequence identity over 169 aligned nucleotides in their core structures, and their encoded proteins show 65.3% amino acid similarities over 219 aligned amino acid positions. This important level of sequence conservation is consistent with our previous finding of closely related group I introns in the chloroplast and mitochondrial genomes of green algae (Turmel et al. 1995; Turmel et al. 1999; Lucas et al. 2001; Turmel, Otis, and Lemieux 2002a) and further supports the idea that lateral transfers of introns have occurred between different organellar compartments during the evolution of green plants.
Repeated Sequence Elements
A comparison of the Pseudendoclonium mtDNA sequence against itself using PIPMAKER (Schwartz et al. 2000) revealed the presence of many degenerated repeated elements within intergenic regions and introns (fig. 3). We identified these repeated sequences using the program REPUTER and found that they are up to 350 nt in size and do not differ substantially from the rest of the Pseudendoclonium genome in terms of nucleotide composition. The repeats that were 50 nt or more were classified using REPEATFINDER (Volfovsky, Haas, and Salzberg 2001); they represent about 10 kb of the genome and form four distinct classes, designated C1 to C4 (fig. 3 and table 5). The members of a given class share no sequence similarity with those of other classes. The first class of repeats (C1) is the largest. Its members map to at least 16 different loci (fig. 3) and can be further classified into 14 subclasses, designated C1R1 through C1R14. The longest repeat within each subclass, defined as the prototype, is made up of repeated units (subrepeats) that also constitute the shorter repeated elements found in the same subclass. Distinct subclasses feature different arrangements of repeated units. The coordinates of the prototypes and number of repeated elements within each subclass are given in table 5. The abundance of subclasses in class 1 suggests that numerous recombination and shuffling events between repeated units took place during the evolution of the mitochondrial genome. On the other hand, classes 2, 3, and 4 are much less complex than class 1. Each exhibits only one subclass of repeats, with two identical copies of the prototype sequence that are far apart from one another. It is likely that these elements arose from single duplication events. In addition to the repeats described above, Pseudendoclonium mtDNA features numerous repeated elements less than 50 nt that span about 4 kb of this genome (fig. 3).
FIG. 3. Positions of repeated sequence elements in Pseudendoclonium mtDNA as revealed by PIPMAKER. Repeats of 50 nt or more were sorted into four classes by REPEATFINDER. Repeats less than 50 nt were not classified. Genes and their polarities are denoted by horizontal arrows, and exons are represented by filled boxes. Similarities between repeated sequence elements are shown as average percentage identity (between 50% to 100% identity)
Table 5 Prototypes of Repeated Sequence Elements of 50 nt or More in Pseudendoclonium mtDNA.
Phylogenetic Analyses
The amino acid sequences derived from the seven protein-coding genes (cob, cox1, nad1, nad2, nad4, nad5, and nad6) that are common to the mtDNAs of Pseudendoclonium and 11 other green plants were concatenated and analyzed with ML and ML-distance methods of phylogenetic inference, using as outgroup the homologous sequences from Mesostigma. Note that when the red algae Porphyra purpurea and Chondrus crispus were used as outgroup, we observed no change in the position of Pseudendoclonium. The WAG-F model of amino acid replacement was selected for all phylogenetic analyses, because it gave higher likelihood values than the JTT-F and mtREV24-F models (table 6). The ML and ML-distance trees inferred by assuming -distributed rates of substitutions across sites revealed a strong affiliation of Pseudendoclonium with the highly supported clade containing Pedinomonas and the four chlorophycean green algae whose mtDNA sequences has been determined to date (fig. 4). As shown by their very long branches, the members of this clade, designated here "reduced-derived" clade, display a higher rate of mtDNA sequence evolution compared to the other green plants examined. Phylogenetic relationships among members of the "reduced-derived" clade were found to be well resolved in all analyses, and the affiliation of Pseudendoclonium with this clade remained strongly supported when we excluded invariable sites and/or when we included an additional parameter (Mgene option in CODEML) to take into account the different evolutionary rates of the selected mitochondrial proteins.
Table 6 Tree-Related Statistics and Log-Likelihood Values for the Mitochondrial Data Set of 2,107 Amino Acid Positions.
FIG. 4. Phylogenetic position of Pseudendoclonium as inferred from the deduced amino acid sequences of seven mitochondrial genes. ML and ML-distances analyses of the data set (2,107 amino acid positions) were carried out under the WAG-F model of amino acid replacement, assuming -distributed rates of substitutions across sites. The best-supported ML tree computed with CODEML is shown. Numbers above the nodes indicate support values; upper and lower values are from the ML and ML-distance analyses, respectively. The chlorophyte lineages displaying a "reduced-derived" pattern of mtDNA evolution are represented as thick lines
To our surprise, we found that under certain conditions of phylogenetic analyses the "reduced-derived" clade groups with the rest of green algae and land plants. For instance, in the best ML tree shown in figure 4, the cluster formed by Pseudendoclonium and the "reduced-derived" clade occupies the Chlorophyta lineage. This result contrasts with previously reported phylogenies in which members of the "reduced-derived" clade branch consistently outside the monophyletic group formed by the rest of green plants, most certainly as a result of long-branch attraction artifacts (Turmel, Otis, and Lemieux 1999; Nedelcu et al. 2000). We explored the phylogenetic conditions that contribute to the recovery of the correct branching pattern in our best ML tree and found that the selected inference method, evolutionary model, and taxon sampling all play an important role in the analyses. Analyses based on ML-distances and performed under the JTT-F and mtREV24-F models failed to recover the correct topology as the best tree. Moreover, the inclusion of the Pseudendoclonium taxon in the concatenated data set and the addition of a correction for amino acid substitutions across sites also represent important factors in the ML analyses. However, even under the most favorable ML conditions for recovering the correct tree, alternate topologies in which the "reduced-derived" cluster branches outside the clade formed by the rest of green plants were detected relatively frequently among the RELL bootstrap replicates. These alternate topologies proved to be not significantly different (P < 0.1) from the best tree in the Kishino-Hasegawa and the Shimodaira-Hasegawa tests.
Rates of Amino Acid Substitutions
Considering the branch lengths of the best ML tree (fig. 4), it appears that Pseudendoclonium mtDNA evolves at a slower rate than the mtDNAs of taxa from the "reduced-derived" clade. To determine whether this interpretation is correct, we assessed the relative rates of amino acid substitutions of mtDNA-encoded proteins among the different lineages using the binomial test of Gu and Li (1992) (table 7). The substitution rates of Pseudendoclonium proteins were found to be significantly smaller (P < 0.001) than those observed for Scenedesmus and other green algae from the "reduced-derived" clade. Pseudendoclonium mtDNA–encoded proteins, however, evolve significantly faster (P < 0.001) than their Nephroselmis and Prototheca counterparts.
Table 7 Differences in the Number of Amino Acid Substitutions in Seven Mitochondrial Proteins and Test for Equal Rates of Substitutions in Selected Pairs of Green Algal Lineages.
Discussion
Distinctive Features of Pseudendoclonium mtDNA
Of all the green-algal mtDNAs sequenced to date, Pseudendoclonium mtDNA is the genome with the largest size and the lowest gene density (table 8). At 95,880 bp, the size of this genome is almost twofold larger than those of the two chlorophytes displaying an ancestral pattern of evolution (Nephroselmis and Prototheca). Despite this substantial expansion in size, Pseudendoclonium mtDNA displays a smaller repertoire of conserved genes compared with its Nephroselmis and Prototheca counterparts (table 8). Most of its increased size is accounted for by dispersed repeats and sequences of unknown nature/origin that reside mainly within the intergenic regions.
Table 8 Compared Features of Pseudendoclonium mtDNA and Other Green-Plant mtDNAs.
The unusually large size and low gene density of Pseudendoclonium mtDNA, together with the presence of dispersed repeats, is reminiscent of the "expanded" pattern of evolution exhibited by embryophyte mtDNAs. Both the expanded Pseudendoclonium and the embryophyte mtDNAs encode a similar number of conserved genes, although they differ in their gene repertoire; moreover, most of their extraneous DNA sequences are of unknown origin and/or function (Unseld et al. 1997; Kubo et al. 2000; Notsu et al. 2002). The finding that Pseudendoclonium mtDNA is much larger than its chlorophyte counterparts suggests that mitochondrial genome expansion occurred independently in the Chlorophyta and Streptophyta. Analyses of additional ulvophyte mtDNAs will be required to determine whether the Ulvophyceae displays the pattern of progressive genome expansion observed in the Streptophyta.
On one hand, the gene content (a relatively complex gene repertoire) and gene structure (lack of fragmented and scrambled rRNA genes) of Pseudendoclonium mtDNA are characteristic of "ancestral" mtDNAs. On the other hand, its low gene density, abundant repeats, and the absence of certain genes (nad9, rpl6, rps7, and rrn5) are typical of Scenedesmus and Pedinomonas mtDNAs. The finding of genomic features that are typical of the "reduced-derived" pattern of mtDNA evolution, together with the presence of ancestral features, suggests that Pseudendoclonium belongs to a lineage that appeared after the emergence of the Trebouxiophyceae but before the divergence of the Chlorophyceae. Further supporting this notion are three independent observations. First, ML and ML-distance trees inferred from mtDNA-encoded proteins always cluster Pseudendoclonium with chlorophycean green algae and consistently place the trebouxiophyte Prototheca at the base of this clade (see next section). Second, the overall rate of sequence evolution appears to be accelerated to an intermediary level in Pseudendoclonium mtDNA as compared with the slow rates observed in "ancestral" mtDNAs and the very fast rates detected in Pedinomonas, Scenedesmus and chlorophycean green-algal mtDNAs (fig. 4). Third, close relatives of the Pacox1.2, Pacox1.4, and Parnl.1 introns are present in mtDNAs displaying both the "ancestral" and "reduced-derived" patterns of evolution.
Phylogenetic Niche of the Ulvophyceae
Our phylogenetic analyses of concatenated mtDNA-encoded protein sequences reveal a close relationship between Pseudendoclonium and chlorophycean green algae, with the trebouxiophyte Prototheca occupying a basal position (fig. 4). These analyses included all the green-algal mtDNA sequences available in public databases to minimize possible undesirable effects of small taxon sampling and used Mesostigma viride as outgroup to maximize the amount of phylogenetic information. The position of Pseudendoclonium relative to the Chlorophyceae and Trebouxiophyceae may be the result of genuine phylogenetic signal because it is consistent with our finding that Pseudendoclonium mtDNA shares derived structural features with its homologs in chlorophycean green algae. However, the branching order of the Chlorophyceae/Ulvophyceae/Trebouxiophyceae remains an unresolved issue. The tree recovered in our analyses may reflect variations in the evolutionary rates of mtDNA sequences rather than the underlying phylogenetic signal, and although we have included all currently available green-algal mtDNAs in our analyses, taxon sampling is still very low, with only one representative of the Ulvophyceae and of the Trebouxiophyceae.
Pseudendoclonium appears to be closely related to Pedinomonas, a green alga of uncertain affiliation. In mitochondrial trees, Pedinomonas branches immediately after the emergence of the Pseudendoclonium lineage. Given the very long branch displayed by Pedinomonas, the phylogenetic position of this chlorophyte might be attributed to long-branch artifacts. However, the existence of a close relationship between Pedinomonas and ulvophytes is supported by the observation that the basal bodies of Pedinomonas and ulvophytes display an absolute counterclockwise orientation that contrasts with the directly opposed or clockwise orientation found in chlorophycean green algae (Melkonian 1990). Considering that the 25-kb mtDNA of Pedinomonas is highly reduced in size and gene content relative to its Pseudendoclonium and Scenedesmus relatives and also considering the much longer branch displayed by Pedinomonas mtDNA, it appears that the mitochondrial genome evolved at a more accelerated rate in the lineage leading to Pedinomonas than in the Pseudendoclonium and Scenedesmus lineages.
Repeated Sequence Elements As an Evolutionary Force in the Chlorophyta
Small repeats have been previously identified in chlorophyte mtDNAs. The few that are found in Prototheca mtDNA vary from 30 to 200 nt, are mostly arranged in tandem, and the recurring motifs are rich in A+T (Wolff et al. 1994). Repeats in Scenedesmus mtDNA range from 16 to 118 nt, and, like their Pseudendoclonium counterparts, account for at least 15% of the genome, flank many individual genes, are composed of various subrepeats, and show a low bias in base composition (Nedelcu et al. 2000). The repeats harbored by chlamydomonad mtDNAs are shorter (9 to 14 nt) and richer in G+C (Boer and Gray 1991; Nedelcu and Lee 1998). Unlike all their known chlorophyte counterparts, the numerous repeated sequences found in Pedinomonas mtDNA are densely packed within a discrete, noncoding region of the genome (Turmel et al. 1999). The low abundance of repeats in Nephroselmis and Prototheca mtDNAs, together with the increased occurrence of these elements in the more derived lineages leading to Pseudendoclonium and Scenedesmus, raise the question on their origin. Repeats might have been present in the mitochondrial genome of the last common ancestor of the ulvophytes and chlorophycean green algae, and have diverged after the split of these lineages. Alternatively, they might have arisen independently in the Ulvophyceae and Chlorophyceae.
Considering that recombination between dispersed repeats can lead to genome rearrangements (gene losses or inversions) and that gene content became reduced as families of dispersed repeats emerged and grew in size during the evolution of chlorophyte mtDNAs (at least in derived lineages), we speculate that repeated elements have played an important role in generating the great diversity of size and gene arrangement seen in these mtDNAs. In chlamydomonad mtDNAs, where gene content is the poorest of the Chlorophyta lineage, excision of coding regions via recombination between flanking short repeats has been invoked to explain their reduced gene content (Nedelcu 1997; Nedelcu 1998). Dispersed repeats are also thought to act as hot spots for recombination in land-plant (Palmer and Herbon 1988; Mackenzie, He, and Lyznik 1994), fungal (Jamet-Vierny, Boulay, and Briand 1997), and animal (Lunt and Hyman 1997) mtDNAs. Aside from dispersed repeats, other factors most probably determine the tempo of gene loss. For angiosperms, it has been shown that the tempo of mitochondrial gene loss (and probably gene transfer to the nucleus) is remarkably punctuated. Certain lineages have rapidly lost most or all of their 16 ribosomal protein and sdh genes, whereas other lineages, mostly ancient lineages, have maintained a constant set of mitochondrial genes for hundreds of millions of years (Adams et al. 2002). This punctuated pattern appears to be driven by major episodic rises in the rate of functional gene transfer.
Interestingly, short, dispersed repeats have been observed in the chloroplast DNAs of Chlamydomonas taxa (Boudreau and Turmel 1996; Maul et al. 2002) and of the trebouxiophyte Chlorella vulgaris (Maul et al. 2002) but not in Nephroselmis chloroplast DNA (Maul et al. 2002). The phylogenetic distribution of small, repeated elements within chlorophyte chloroplast DNAs thus parallels that observed for the mitochondrial genome. Given the evidence for lateral transfers of introns (genetic elements often associated with short repeats) between the mitochondrial and chloroplast compartments (Turmel et al. 1995; Turmel et al. 1999; Lucas et al. 2001; Turmel, Otis, and Lemieux 2002a), it is possible that such events contribute to the dispersal of short repeats and account for the presence of these elements in both organelle genomes of the same cell. In this context, it will be interesting to see if short, repeated elements are found in ulvophyte chloroplast DNAs.
Conclusion
Representing the first ulvophyte organelle genome sequence publicly available, Pseudendoclonium mtDNA is a valuable source of information to our understanding of mitochondrial genome evolution in the Chlorophyta. It will be important to examine mtDNAs from additional representatives of the Ulvophyceae to determine if the distinctive traits exhibited by Pseudendoclonium mtDNA are conserved in other ulvophyte mtDNAs or are restricted to the lineage leading to this ulvophyte. A broader study of mitochondrial genomes, including more trebouxiophytes and chlorophycean green algae, will be required to resolve the question about the monophyly of the Ulvophyceae, to clarify the interrelationships between the Chlorophyceae, Trebouxiophyceae, and Ulvophyceae, and also to better understand how the great diversity seen at the mitochondrial genome level arose during the evolution of chlorophytes.
Supplementary Material
The genome sequence reported in this paper has been deposited in the GenBank database (accession number AY359242).
Acknowledgements
This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (to M.T. and C.L.). J.-F.P. gratefully acknowledges a scholarship from CREFSIP (Centre de Recherche sur la Fonction, la Structure et l'Ingénierie des Protéines).
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