The Chloroplast Genome Sequence of the Green Alga Pseudendoclonium akinetum (Ulvophyceae) Reveals Unusual Structural Features and New Insigh
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分子生物学进展 2005年第9期
Département de biochimie et de microbiologie, Université Laval, Québec G1K 7P4, Canada
E-mail: monique.turmel@rsvs.ulaval.ca.
Abstract
One major lineage of green plants, the Chlorophyta, is represented by the green algal classes Prasinophyceae, Ulvophyceae, Trebouxiophyceae, and Chlorophyceae. The Prasinophyceae occupies the most basal position in the Chlorophyta, but the branching order of the Ulvophyceae, Trebouxiophyceae, and Chlorophyceae remains unresolved. The chloroplast genome sequences currently available for representatives of three chlorophyte classes have revealed that this genome is highly plastic, with Chlamydomonas (Chlorophyceae) and Chlorella (Trebouxiophyceae) showing fewer ancestral features than Nephroselmis (Prasinophyceae). We report the 195,867-bp chloroplast DNA (cpDNA) sequence of Pseudendoclonium akinetum (Ulvophyceae), a member of the class that has not been previously examined for detailed cpDNA analysis. This genome shares common evolutionary trends with its Chlorella and Chlamydomonas homologs. The gene content, number of ancestral gene clusters, and abundance of short dispersed repeats in Pseudendoclonium cpDNA are intermediate between those observed for Chlorella and Chlamydomonas cpDNAs. Although Pseudendoclonium cpDNA features a large inverted repeat, its quadripartite structure is unusual in displaying an rRNA operon transcribed toward the large single-copy (LSC) region and a small single-copy region containing 14 genes that are normally found in the LSC region. Twenty-seven group I introns lie in nine genes and fall within four subgroups (IA1, IA2, IA3, and IB); 19 encode putative homing endonucleases, and 7 have homologs at identical insertion sites in other chlorophyte or streptophyte organelle genomes. The high similarity observed among the 14 IA1 and 7 IA2 introns and their encoded endonucleases suggests that many introns arose from intragenomic proliferation of a few founding introns in the lineage leading to Pseudendoclonium. Interestingly, one intron (in atpA) and some of the dispersed repeats also reside in Pseudendoclonium mitochondria, providing strong evidence for interorganellar lateral transfer of these genetic elements. Phylogenetic analyses of 58 cpDNA-encoded proteins and genes support the hypothesis that the Ulvophyceae is sister to the Trebouxiophyceae but cannot eliminate the hypothesis that the Ulvophyceae is sister to the Chlorophyceae. We favor the latter hypothesis because it is strongly supported by phylogenetic analyses of gene order data and by independent structural evidence based on shared gene losses and rearrangement break points within ancestrally conserved gene clusters.
Key Words: green algae ? Ulvophyceae ? Pseudendoclonium akinetum ? chloroplast genome evolution ? group I introns ? repeated sequences
Introduction
The green algae are divided into the phyla Streptophyta and Chlorophyta. The Streptophyta (Bremer 1985) contains all land plants and the green algae belonging to the class Charophyceae (Graham, Cook, and Busse 2000), whereas the Chlorophyta (Sluiman 1985) contains virtually all of the other green algae, i.e., the members of the classes Prasinophyceae, Ulvophyceae, Trebouxiophyceae, and Chlorophyceae (Lewis and McCourt 2004). The basal position of the Prasinophyceae in the Chlorophyta is generally well established, but the branching order of the Ulvophyceae, Trebouxiophyceae, and Chlorophyceae (UTC) remains unresolved (Friedl and O'Kelly 2002; Pombert et al. 2004). A third lineage at the base of the Streptophyta and Chlorophyta is possibly represented by Mesostigma viride; however, some studies suggest that this green alga traditionally classified within the Prasinophyceae represents a basal divergence within the Streptophyta (Bhattacharya et al. 1998; Marin and Melkonian 1999; Karol et al. 2001).
To improve our understanding of phylogenetic relationships among green algae and also to better understand how the chloroplast genome has evolved in this algal group, we have undertaken the complete sequencing of chloroplast DNA (cpDNA) from representatives of various lineages in the Streptophyta and Chlorophyta. Five green algal chloroplast genome sequences are currently available in public databases. The genomes of the prasinophyte Mesostigma (Lemieux, Otis, and Turmel 2000) and of the charophyte Chaetosphaeridium globosum (Turmel, Otis, and Lemieux 2002) most closely resemble their land plant counterparts in terms of overall structure and gene organization. Like most land plant cpDNAs, they feature a quadripartite structure that is characterized by the presence of two copies of an rRNA-encoding inverted repeat (IR) sequence separating a small single-copy (SSC) and a large single-copy (LSC) region. In the Streptophyta, each of these genomic regions shows a highly conserved gene content, and the rRNA operon within the IR is transcribed toward the SSC region.
In contrast, the complete cpDNA sequences of the prasinophyte Nephroselmis olivacea (Turmel, Otis, and Lemieux 1999), the trebouxiophyte Chlorella vulgaris (Wakasugi et al. 1997), and the chlorophycean alga Chlamydomonas reinhardtii (Maul et al. 2002) indicate that the architecture of the chloroplast genome is very fluid in the Chlorophyta. Nephroselmis cpDNA displays ancestral gene clusters and the typical quadripartite structure observed in the streptophytes, whereas Chlorella cpDNA lacks the IR as well as several genes and has gained three group I introns. Chlamydomonas cpDNA is even more scrambled in its structure and gene organization. Although it features an IR, the two single-copy regions are about equal in size and each includes genes usually present in the SSC and LSC regions. Chlamydomonas cpDNA carries fewer genes but more introns than its Chlorella homolog; moreover, it harbors fragmented ancestral operons, and the coding regions of some genes are greatly expanded or broken relative to the corresponding genes in other completely sequenced cpDNAs. Many repeated sequence elements (Maul et al. 2002) are dispersed throughout both the Chlamydomonas and Chlorella genomes; however, such repeats have not been identified in Nephroselmis, Mesostigma, and Chaetosphaeridium cpDNAs.
Here we report the chloroplast genome sequence of Pseudendoclonium akinetum, a unicellular green alga thought to belong to a deep-branching lineage within the Ulvophyceae (Floyd and O'Kelly 1990). This genome has a unique architecture, although it shares some features with Chlorella cpDNA and others with Chlamydomonas cpDNA. Based on our phylogenetic inferences from chloroplast sequences and gene order data as well as on independent structural evidence, we strongly favor the hypothesis that the Ulvophyceae and Chlorophyceae are sister groups.
Materials and Methods
DNA Isolation and Sequencing
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. An A+T–rich fraction containing cpDNA and mitochondrial DNA (mtDNA) was isolated and sequenced as described previously (Pombert et al. 2004). The nuclear-encoded 18S rRNA gene was amplified by polymerase chain reaction (PCR) from total cellular DNA using primers NS1 (White et al. 1990) and 18L (Hamby et al. 1988) as described in Helms et al. (2001). Sequences were edited and assembled with SEQUENCHER 4.2.1 (Gene Codes, Ann Arbor, Mich.).
Sequence Analyses
Genes were identified by Blast homology searches (Altschul et al. 1990) against the nonredundant database of the National Center for Biotechnology Information (NCBI) server (http://www.ncbi.nlm.nih.gov/BLAST/). Positions of open reading frames (ORFs) and protein-coding genes were determined using ORFFINDER at NCBI and various programs of the GCG Wisconsin package (version 10.2) (Accelrys, Burlington, Mass.), whereas genes coding for tRNAs were localized with tRNAscan-SE 1.23 (Lowe and Eddy 1997). Patterns of codon usage for protein-coding genes and ORFs were compared using the CORRESPOND and CODONPREFERENCE programs of the GCG package and the CAI program of the EMBOSS package (version 2.6.0) (http://emboss.sourceforge.net). Repeated sequences were identified with PipMaker (Schwartz et al. 2000) and REPuter 2.74 (Kurtz et al. 2001). Repeats were sorted with REPEATFINDER (Volfovsky, Haas, and Salzberg 2001), and the retrieved classification was refined manually. Putative stem-loop structures and degenerated repeats were identified with PALINDROME and ETANDEM, respectively; these two programs are part of the EMBOSS package. Homologous introns were identified by BlastN searches (Altschul et al. 1990) against the nonredundant database of NCBI using an E value threshold of 1 x 10–6. Homologous introns inserted at identical positions within the same gene were identified by manual screening of the GOBASE database (O'Brien et al. 2003).
Phylogenetic Analyses
The 18S rDNA sequence alignment of Friedl and O'Kelly (2002) was retrieved from TreeBASE (http://www.treebase.org/treebase/), modified to include the P. akinetum sequence, and analyzed as described by these authors. To obtain the chloroplast amino acid data set, sequences of individual chloroplast proteins were aligned using T-COFFEE 1.37 (Notredame, Higgins, and Heringa 2000), the ambiguously aligned regions of these alignments were removed with GBLOCKS 0.91b (Castresana 2000), and the filtered sequences were concatenated. The nucleotide data set was generated by aligning the sequences of individual protein-coding genes on the basis of corresponding protein alignments, filtering the alignments with GBLOCKS 0.91b, concatenating the filtered sequences, and subsequently removing third codon positions with PAUP* 4.0b10 (Swofford 2002). Maximum likelihood (ML) trees inferred from amino acid sequences were computed with PHYML 2.4.4 (Guindon and Gascuel 2003) under the cpREV45 + model (Adachi et al. 2000), whereas ML trees inferred from nucleotide sequences were computed with PAUP* 4.0b10 (Swofford 2002) under the general time reversible + + I model. Modeltest 3.6 (Posada and Crandall 1998) identified the latter model as the one best fitting our nucleotide data. Bootstrap support for each node was calculated using 100 replicates. The confidence limits of alternative tree topologies were evaluated using the Shimodaira-Hasegawa test as implemented in CODEML 3.14 (Yang 1997) and PAUP* 4.0b10 (Swofford 2002). Support for the T1, T2, and T3 topologies by individual proteins in the amino acid data set was estimated with CODEML 3.14 using the MGENE = 1 option.
Intron sequences were aligned manually on the basis of secondary structure predictions, and regions judged to be ambiguously aligned were removed. Neighbor-Joining analyses of the resulting data sets were carried out with PAUP* 4.0b10 (Swofford 2002) using Hasegawa-Kishino-Yano 85 distances with 1,000 bootstrap replicates.
Phylogenetic reconstructions from gene order data were performed with GRAPPA 2.0 (Moret et al. 2001) using the default parameters and the –m (tighter circular lower bound) option. Because this program requires that all genes analyzed be shared between the taxa examined, the genes present within copy B of the IR were excluded from the data set to accommodate the lack of the IR in Chlorella cpDNA.
Results
Phylogenetic Affiliation of P. akinetum
Because it has not been previously demonstrated that P. akinetum (UTEX 1912) belongs to the Ulvophyceae, we have ascertained this affiliation by conducting phylogenetic analyses of nuclear-encoded 18S rDNA sequences with ML, maximum parsimony, and distance methods (Supplementary Fig. S1, Supplementary Material online). Pseudendoclonium akinetum was found to clearly affiliate with other members of the Ulvophyceae, being part of a clade including Pseudendoclonium basiliense and five other members of the Ulotrichales. The P. akinetum and P. basiliense 18S rDNA sequences differ at only seven positions in the data set analyzed.
Genome Structure and Gene Partitioning
Pseudendoclonium cpDNA (195,867 bp) contains two copies of an IR sequence that are separated from one another by LSC and SSC regions (table 1 and fig. 1). The Pseudendoclonium IR encodes only the rRNA operon, in contrast to the IRs of all previously sequenced green plant cpDNAs that include additional genes. Surprisingly, the Pseudendoclonium rRNA operon is transcribed toward the LSC region rather than toward the SSC region. Another unusual feature of Pseudendoclonium cpDNA concerns the pattern of gene partitioning. The SSC region of this genome features 14 genes that map to the LSC region in Mesostigma, Nephroselmis, and land plant cpDNAs (fig. 1). However, the Pseudendoclonium LSC region contains no genes that are usually found in the SSC region.
Table 1 Compared Features of Pseudendoclonium and Other Green Algal cpDNAs
FIG. 1.— Gene map of Pseudendoclonium cpDNA. Genes outside the map are transcribed clockwise. The transcription direction of the rRNA operons is indicated by arrows. The genes shown in yellow, blue, and red map to the IR, LSC, and SSC regions in Mesostigma cpDNA, respectively. Genes and ORFs absent from Mesostigma cpDNA are shown in gray. Gene clusters shared specifically with Chlorella are denoted by thick brackets, whereas the clusters shared with Chlorella and one or more additional chlorophytes are represented by thin brackets. Only the free-standing ORFs larger than 100 codons are shown. tRNA genes are indicated by the one-letter amino acid code followed by the anticodon in parentheses (Me, elongator methionine; Mf, initiator methionine).
Gene Content and Gene Density
Table 2 compares the gene content of Pseudendoclonium cpDNA with those of other green algal cpDNAs. It can be seen that a large number of genes found in both Mesostigma and Nephroselmis (including all ndh genes) have been lost before the emergence of the UTC lineages. With its 105 genes, the chloroplast gene repertoire of Pseudendoclonium is intermediate in size between those of Chlorella and Chlamydomonas (see table 1). Eight of the genes identified in Chlorella (chlB, chlL, chlN, cysA, cysT, trnL(gag), trnS(gga), and trnT(ggu)) are absent from Pseudendoclonium, whereas all of the genes found in Pseudendoclonium except trnR(ccu) are present in Chlorella. Fourteen of the genes identified in Pseudendoclonium (accD, chlI, infA, minD, psaI, psaM, rpl12, rpl19, rpl32, ycf20, ycf62, trnL(caa), trnR(ccg), and trnR(ccu)) are absent from Chlamydomonas, whereas all of the genes found in Chlamydomonas except chlB, chlL, and chlN are present in Pseudendoclonium.
Table 2 Gene Content in Pseudendoclonium and Other Green Algal cpDNAs
A total of 19 of the 27 introns displayed by Pseudendoclonium cpDNA carry an internal ORF encoding a putative homing endonuclease (see table 4). In addition to these intron ORFs, 12 free-standing ORFs larger than 100 codons are present in Pseudendoclonium cpDNA; only 2 (orf286 and orf521) show a codon usage not significantly different from that observed for the protein-coding genes.
Table 4 Group I Introns in Pseudendoclonium cpDNA
Genes are less tightly packed in Pseudendoclonium cpDNA than in Mesostigma and Nephroselmis cpDNAs (table 1). The intergenic spacers in this ulvophyte genome are up to 3,215 bp in size, with an average of 600 bp. The higher proportion of coding sequences found in Pseudendoclonium cpDNA relative to Chlorella cpDNA is explained by the greater number of introns in the former genome (table 1).
Expansion of Coding Regions
Eight protein-coding genes in Pseudendoclonium cpDNA are nearly two or three times larger than their Mesostigma homologs (table 3). For all of these genes, with the exception of cemA, expansion of coding regions appears to be the main cause of their increased sizes. Because the sizes of the corresponding Mesostigma genes match those of their counterparts in streptophytes and the cyanobacterium Synechocystis sp. PCC 6803, we eliminated the alternative hypothesis that shrinkage of the Mesostigma coding regions is responsible for the observed size variations. The ftsH, rpoB, rpoC1, and rpoC2 genes represent the most notable cases of gene expansion; the levels of expansion noted for these genes are comparable or greater than those for the corresponding genes in Chlamydomonas (table 3). The extra sequences accounting for the expansion of the Pseudendoclonium ftsH and rpo genes map mainly to internal coding regions. In Chlamydomonas, rpoB, rpoC1, as well as rps2 each consist of two separate ORFs, and even though these ORFs are adjacent, no intron has been detected in the noncoding sequence separating them. In Pseudendoclonium, each of these three genes occurs as a single ORF.
Table 3 Compared Sizes of Expanded Genes in Pseudendoclonium, Chlorella, and Chlamydomonas cpDNAs
Introns
The 27 introns in Pseudendoclonium cpDNA account for 14.8% of the genome size and are preferentially located in genes coding for photosynthetic proteins (table 4). They all belong to the group I family and fall within subgroups IA1, IA2, IA3, and IB according to the classification system of Michel and Westhof (1990). The subgroup IA1 is well represented by the Pseudendoclonium introns, with 14 introns assigned to this category.
Structural and phylogenetic analyses of the IA1 and IA2 introns revealed close relationships among members of the same subgroup. These introns are well conserved in both primary sequences and secondary structures (fig. 2A, B, C, and D); moreover, those sharing similar ORFs tend to cluster together in phylogenetic trees inferred from core sequences (compare fig. 2E with fig. 2C and D). The seven IA1 intron-encoded H-N-H endonucleases are all specified by an ORF in L5 (table 4) and can be assigned to three categories based on sequence similarity within the region containing the H-N-H motif (fig. 2E). Pa.psbA.1 differs from the other IA1 introns in carrying an ORF in L9 that potentially codes for an endonuclease of the LAGLIDADG family (fig. 2E). Five IA2 introns exhibit an ORF in L6 that codes for a putative GIY-YIG endonuclease; these IA2 intron-encoded proteins can be divided into two categories based on sequence similarity (fig. 2D and E).
FIG. 2.— Comparative analyses of group I introns and intron ORFs in Pseudendoclonium cpDNA. (A and B) Consensus secondary structure models of introns displayed according to Burke et al. (1987): (A) IA1 introns and (B) IA2 introns. Arrows denote the 5' splice sites between exon and intron residues. Highly conserved residues (in 11 IA1 and 5 IA2 introns) and slightly less conserved residues (in nine IA1 and four IA2 introns) are shown in uppercase and lowercase characters, respectively; the other residues are represented by filled circles. Conserved base pairings (in 11 IA1 and 6 IA2 introns) are denoted by bars. Numbers inside the variable loops indicate the size variations of these loops in the compared introns. Note that the P2 pairing is missing in one IA1 intron (Pa.psbA.7). (C and D) Neighbor-Joining analyses of intron core sequences: (C) IA1 introns and (D) IA2/IA3 introns. The Chlamydomonas reinhardtii (Cr) and Chlamydomonas eugametos (Ce) introns homologous to the Pseudendoclonium (Pa) introns (see table 5) were included in these analyses. Nodes that were identified in at least 80% of the bootstrap replicates are labeled with asterisks. The introns denoted in green, red, blue, and orange encode putative homing endonucleases; in each panel, those denoted by the same color specify closely related proteins. (E) Alignment of the endonuclease regions containing the H-N-H, LAGLIDADG, or GIY-YIG motif. The amino acid residues making up each motif are underlined.
Table 5 Group I Introns at Identical Gene Locations in Pseudendoclonium cpDNA, Other Green Algal cpDNAs, and Land Plant cpDNAs
The IA3 and IB introns show more sequence divergence than their IA1 and IA2 counterparts. As shown in Supplementary Figure S2 (Supplementary Material online) and table 4, the two IA3 introns (Pa.psbA.3 and Pa.rrl.1) differ greatly with respect to their core structures and encoded endonucleases. The P3.1 and P3.2 pairings are present in Pa.psbA.3 but not in Pa.rrl.1, and Pa.psbA.3 encodes a putative GIY-YIG endonuclease, whereas Pa.rrl.1 encodes a putative endonuclease with a single LAGLIDADG motif (table 4). Each of the four IB introns contains an ORF in L8 that codes for a putative LAGLIDADG endonuclease with one or two copies of this motif (table 4); however, these ORFs are distantly related in sequence.
Our BlastN searches of the GenBank database for homologs of the Pseudendoclonium chloroplast introns identified only introns from green algae and land plants (E value threshold of 1x 10–6). The most surprising result of this analysis was the finding of homologous introns (Pa.atpA.1 and Pa.atp1.1) inserted at the same gene position in the chloroplast and mitochondrial genomes of Pseudendoclonium (fig. 3). In addition to highly similar primary sequences and secondary structures, these introns feature in L8 very similar ORFs encoding putative endonucleases with a double LAGLIDADG motif.
FIG. 3.— Comparative analyses of the chloroplast Pa.atpA.1 and mitochondrial Pa.atp1.1 introns. (A) Consensus secondary structure models of the two introns displayed according to Burke et al. (1987). The arrow denotes the 5' splice site between the exon and intron residues. Identical residues are shown in uppercase characters, whereas different residues are represented by filled circles. Conserved base pairings are denoted by bars. Upper and lower numbers inside the variable loops indicate size variations in the Pa.atpA.1 and Pa.atp1.1 introns, respectively. (B) ClustalW alignment of the putative LAGLIDADG endonucleases encoded by the two introns. The LAGLIDADG motifs are underlined.
Table 5 reports the homologous group I introns of chloroplast origin that proved to be inserted at the same sites as their Pseudendoclonium counterparts. Five Pseudendoclonium introns have known homologs in chlorophycean green algae, whereas homologs of Pa.rrl.1 have been observed in prasinophytes, trebouxiophytes, chlorophycean green algae, and the hornwort Anthoceros punctatus. The three psbA and rrl introns in C. reinhardtii cpDNA exhibit a high degree of primary sequence and secondary structure conservation with their Pseudendoclonium counterparts (Supplementary Fig. S2, Supplementary Material online). Even the unusual P3.1 and P3.2 pairings found in Pa.psbA.3 are present in Cr.psbA.2.
It is intriguing that L9 of Pa.psbA.1 codes for a putative single LAGLIDADG endonuclease and that no endonuclease motif has been assigned to the ORF of 102 codons found within the same loop in the homologous Cr.psbA.1 IA1 intron (Holloway, Deshpande, and Herrin 1999). Our analysis of the published Chlamydomonas cpDNA sequence mapping at this locus revealed the presence of an ORF of 46 codons encoding a LAGLIDADG motif 29 nt upstream of orf102; interestingly, the predicted protein sequence of this ORF exhibits high similarity with the N-terminal domain of the Pa.psbA.1-encoded endonuclease (fig. 2E). To test the possibility that there is a frameshift in the sequence originally published by Holloway, Deshpande, and Herrin (1999), we sequenced a PCR product containing the Cr.psbA.1 intron; however, we found no nucleotide difference. We conclude that a nonsense mutation led to loss of the LAGLIDADG motif in the Cr.psbA.1-encoded protein.
Repeated Elements
The Pseudendoclonium chloroplast genome contains a large number of repeated elements (fig. 4). Two types of repeated sequences can be distinguished: short tandem repeats and short dispersed repeats (SDRs). The short tandem repeats are found in the vicinity of trnMe(cau) in a region spanning about 2 kb. The units forming this repeat region are 10–20 bp in size, rich in A + T, and degenerated in sequence.
FIG. 4.— PipMaker analysis of Pseudendoclonium cpDNA. This genome sequence was aligned against itself. At the top of the alignment, genes and their polarities are denoted by horizontal arrows, and coding sequences are represented by filled boxes. The SDR repeat units described in table 6 are denoted by boxed letters. The short tandem repeats near trnMe(cau) are shown as gray dots. Similarities between aligned regions are shown as average percent identity between 50% and 100% identity.
Table 6 SDR Repeat Units in Pseudendoclonium cpDNA and mtDNA
The SDRs map to intergenic spacers and/or introns and can be classified into four groups of repeat units (A, B, C, and D) on the basis of their primary sequences (table 6). They occur frequently as palindromic sequences separated by 7–8 bp, and the stem-loop structures often contain more than one SDR unit. As deletion of palindromic structures and other repeated elements is known to occur during cloning in Escherichia coli, we confirmed by direct sequencing of PCR products the sizes and sequences of the Pseudendoclonium SDRs that we identified by analysis of cloned fragments. To our surprise, we found that numerous SDRs in Pseudendoclonium cpDNA bear identical or close sequence similarity to those present in the mitochondrial genome of this alga (Pombert et al. 2004). These homologous mitochondrial SDRs fall within three of the four above-mentioned groups of repeat units (A, B, and C) (table 6) and also occur as stem-loop structures composed of one or more repeat units. To our knowledge, the presence of closely related SDRs in different organelles of the same eukaryotic cell has not been previously reported.
Genome Organization
Our pairwise comparisons of Pseudendoclonium cpDNA with previously sequenced green plant cpDNAs indicate that its gene organization is most similar to that of Chlorella. These two green algal genomes share 16 distinct gene clusters, 3 of which (rps18-rpl20, psbK-ycf12-psaM, and trnE(uuc)-trnMf(cau)) have not been identified in any other sequenced chloroplast genomes (fig. 1). A single gene cluster (trnC(gca)-trnT(ugu)) is shared specifically between Pseudendoclonium and Chlamydomonas cpDNAs.
Twelve of the gene clusters that are conserved between Mesostigma and Nephroselmis cpDNAs have been broken in Pseudendoclonium cpDNA (fig. 5). As revealed by our analysis of the rearrangement break points within these ancestrally conserved clusters, the Pseudendoclonium genome shares 9 of the 10 break points found in Chlorella cpDNA and features 13 additional break points (fig. 5). All of these nine break points are also observed in Chlamydomonas cpDNA (fig. 5). Of the 33 break points exhibited by this chlamydomonad genome, 11 are specifically shared with Pseudendoclonium, whereas a single one is specifically shared with Chlorella. The psbB-psbT-psbN-psbH cluster displays only two break points that are unique to Pseudendoclonium. Although the psbN gene of this ulvophyte has retained its location between psbT and psbH, it has been relocated to the DNA strand encoding the other psb genes (fig. 1).
FIG. 5.— Fragmented ancestral gene clusters in Pseudendoclonium, Chlorella, and Chlamydomonas cpDNAs. The indicated clusters are found in both Mesostigma and Nephroselmis cpDNAs. Note that rpl22 has not been represented in the large ribosomal protein gene cluster; this gene is found in Mesostigma between rps19 and rps3 but is missing from Nephroselmis and the three other chlorophytes. Sites of fragmentation are denoted by arrowheads above the clusters, with the arrowheads at the lower, middle, and upper positions pointing to sites in Chlamydomonas, Pseudendoclonium, and Chlorella, respectively. Genes missing from Chlamydomonas, Pseudendoclonium, and Chlorella are denoted by circles, asterisks, and double dagger, respectively, below the clusters. Gene polarities are not shown.
Phylogenetic Analyses
The amino acid and nucleotide sequences derived from the 58 protein-coding genes (see Supplementary Table S1, Supplementary Material online) that are shared between the cpDNAs of Pseudendoclonium, Chaetosphaeridium, Chlamydomonas, Chlorella, Marchantia, Nephroselmis, and Nicotiana were concatenated and analyzed with ML inference methods using the homologous sequences of Mesostigma as the outgroup. As expected, analyses of both the amino acid (11,225 positions) and nucleotide (25,318 positions) data sets yielded a best tree (T1) in which the chlorophytes and streptophytes form two distinct lineages (fig. 6). In the chlorophyte lineage, the prasinophyte Nephroselmis occupies the most basal position, whereas the trebouxiophyte Chlorella, the ulvophyte Pseudendoclonium, and chlorophycean alga Chlamydomonas form a clade in which Pseudendoclonium is sister to Chlorella. Topology T1 accounted for 91% and 80% of the bootstrap replicates in the analyses of the amino acid and nucleotide data sets, respectively, and was also found to be the most highly supported topology in distance (ML and LogDet distances) and maximum parsimony analyses (data not shown). In all analyses, instability in the branching order of taxa was observed only for the UTC clade. The alternative topology showing Pseudendoclonium as sister to Chlamydomonas (T2) was recovered in 9% and 20% of the bootstrap replicates in the ML analyses of the amino acid and nucleotide data sets, respectively, whereas the topology placing Pseudendoclonium at a basal position in the UTC clade (T3) was not detected in any of the 100 bootstrap replicates. The confidence levels of these alternative topologies were assessed using the statistical test of Shimodaira-Hasegawa. T3, but not T2, proved to be significantly worse than T1 at P < 0.05 in the analyses of both the amino acid and nucleotide data sets (fig. 7).
FIG. 6.— Phylogenetic position of Pseudendoclonium as inferred by ML analyses of 58 cpDNA-encoded proteins and genes. (A) Best protein tree. (B) Best gene tree. Bootstrap values are indicated on the corresponding nodes, and branch lengths are drawn to scale.
FIG. 7.— Evaluation of the three phylogenetic hypotheses for the branching order of UTC taxa using the Shimodaira-Hasegawa test and structural evidence based on shared gene losses and rearrangement break points within ancestrally conserved gene clusters. Values above and below the nodes indicate the number of break points and gene losses, respectively. The P values obtained in the Shimodaira-Hasegawa test are indicated for the ML analyses of both the amino acid (aa) and nucleotide (nt) data sets.
Separate ML analyses of the amino acid data set with CODEML revealed no strong disagreement among the phylogenetic signals provided by the individual proteins (Supplementary Table S1, Supplementary Material online). Topologies T1, T2, and T3 were found to be supported by 22, 25, and 11 proteins, respectively, with RELL bootstrap values ranging from 39% to 93%. However, all 58 proteins, with the exception of rpoB, failed to provide a signal of sufficient strength to reject one or both of the alternative topologies at P < 0.05 in the Shimodaira-Hasegawa test. rpoB supported T1 and rejected T3 (but not T2) at P = 0.02. Thus, it appears that general homoplasy throughout the data set is the most likely explanation for the lack of resolution between T1 and T2.
Phylogenetic relationships were also inferred from gene order data using the relative positions of the 80 chloroplast genes shared by Pseudendoclonium and the seven green plants mentioned above. GRAPPA 2.0, the program used for these analyses, reconstructs phylogenies by assuming that gene rearrangements occur by inversions. The best tree was found to display the T2 topology and to feature a total length of 240 inversion steps (fig. 8). User-tree analyses constrained to the T1 and T3 topologies yielded trees with 6 and 10 extra steps, respectively.
FIG. 8.— Phylogenetic position of Pseudendoclonium as inferred by gene order analysis of 80 chloroplast genes. The best tree is shown with branch lengths drawn to scale.
Mapping of shared gene losses and rearrangement break points located within ancestrally conserved gene clusters on topologies T1, T2, and T3 also revealed that these two sets of structural characters independently support topology T2 as the most parsimonious scenario (fig. 7). A total of 36 rearrangement break points and 41 gene losses were mapped on T2 compared to 47 break points and 46 gene losses on T1 and 46 break points and 45 gene losses on T3. In contrast to T1 and T3, T2 shows several rearrangement events and gene losses that are specifically shared between sister taxa in the UTC lineage.
Discussion
Unusual Features of Pseudendoclonium cpDNA
Like the chloroplast genomes of the chlorophycean green algae belonging to the genus Chlamydomonas, Pseudendoclonium cpDNA is unusual with respect to its quadripartite structure (fig. 1). Fourteen of the genes found in the SSC region of this ulvophyte genome map to a different genomic region (most often the LSC region) in all streptophytes featuring an IR as well as in the prasinophytes Nephroselmis and Mesostigma. Moreover, the Pseudendoclonium IR features an rRNA operon that is transcribed toward the LSC region. During the evolution of ulvophytes, the transfer of genes from the LSC to the SSC region may have been closely associated with the inversion of the IR. In this context, it may be envisioned that an ancestral genome with a conventional quadripartite structure has undergone two consecutive inversions, each involving a segment encompassing the IR and part of the LSC or, alternatively, one inversion involving a segment encompassing the entire IR sequence and the other the second IR sequence. Whatever the mechanism, additional ulvophyte chloroplast genomes will need to be examined to determine whether the atypical quadripartite structure reported here for Pseudendoclonium cpDNA is commonly found in the Ulvophyceae. Prior to our study, the only ulvophyte chloroplast genome that had been investigated by physical and gene mapping is that of Codium fragile, a green alga thought to belong to a later-diverging lineage than the Pseudendoclonium lineage (Manhart et al. 1989). Considering that Codium cpDNA is highly reduced in size (89 kb), lacks an IR, and has a broken rRNA operon, the chloroplast genome appears to have evolved under relaxed constraints in the Ulvophyceae.
Another unusual feature of Pseudendoclonium cpDNA concerns its intron composition (table 4). Of all the chloroplast genome sequences reported so far, this chlorophyte genome is the richest in group I introns. The striking similarity among the 14 IA1 and 7 IA2 introns and the putative homing endonucleases encoded by these introns (fig. 2) suggests that many of the 27 group I introns found in Pseudendoclonium cpDNA arose from intragenomic proliferation of a few founding introns in the lineage leading to Pseudendoclonium. The possibility that numerous Pseudendoclonium introns took their origin from independent invasions of closely related introns by lateral transfer rather than by intragenomic proliferation seems less likely but cannot be excluded. Interestingly, the intron composition of Pseudendoclonium cpDNA resembles that of its mitochondrial counterpart (Pombert et al. 2004) in featuring exclusively group I introns, a substantial fraction of which specifies potential homing endonucleases.
The highly similar group IB introns sharing the same insertion site in the Pseudendoclonium chloroplast atpA and mitochondrial atp1 genes (fig. 3) as well as the closely related SDRs in both organelle DNAs of this green alga (table 6) provide compelling evidence for interorganellar, lateral transfer of these genetic elements. The lateral transfer of the atpA/atp1 introns appears to have occurred specifically in the ulvophyte lineage, considering that these introns represent the first examples of intron insertion at their cognate site in atpA/atp1. Cases of interorganellar transfers of group I introns have been previously documented for green algae (Turmel, Mercier, and C?té 1993; Turmel et al. 1995, 1999); however, the rDNA introns associated with these transfers exhibit a sporadic distribution pattern and occur in more than one chlorophyte lineage, making it difficult to estimate the timing of the lateral transfer event(s).
The cpDNAs of Pseudendoclonium and Other Advanced Chlorophytes Share Common Evolutionary Trends
A number of shared derived features between the cpDNAs of Pseudendoclonium and other advanced chlorophytes highlight common evolutionary trends among the members of the UTC clade. Like its Pseudendoclonium homolog, the completely sequenced cpDNA of the chlorophycean green alga C. reinhardtii as well as other chlamydomonad cpDNAs that have been analyzed by physical and gene mapping display an atypical quadripartite structure in which gene partitioning among the two single-copy regions differs from the conserved pattern observed in all other green plant cpDNAs containing an IR (Boudreau, Otis, and Turmel 1994; Boudreau and Turmel 1995, 1996; Maul et al. 2002). The single-copy regions of these genomes are about equal in size, and their genes have been so extensively shuffled that it is impossible to trace the events that led to their relocations and to determine if the rRNA operons have been altered in their orientation. On the other hand, studies of C. vulgaris and Chlorella ellipsoidea cpDNAs clearly indicate that the IR was the subject of rearrangement or deletion during the evolution of trebouxiophytes (Yamada 1991; Wakasugi et al. 1997). Given the absence of the IR in C. vulgaris and the fragmentary information currently available for the IR-containing cpDNA of C. ellipsoidea, it remains unknown if genes were relocated from one single-copy region to the other. Unlike its C. vulgaris homolog which is intact, the C. ellipsoidea rRNA operon is fragmented into two pieces, one containing the rrs and trnI(gau) genes and the other containing the trnA(ugc), rrl, and rrf genes (Yamada and Shimaji 1987). The orientation of these genes is such that a single inversion of the latter fragment would recreate an intact rRNA operon that is transcribed toward the LSC region. From these data alone, one cannot conclude about the timing of the event(s) that led to the change in orientation of the Pseudendoclonium and C. ellipsoidea rRNA operons. It is possible that the chloroplast genome of the last common ancestor of the UTC algae had already acquired an IR with an rRNA operon transcribed toward the LSC region and an SSC region with some genes usually present in the opposite single-copy region.
UTC algae also exhibit common features at the levels of gene content, gene order, repeated sequences, and intron content. Pseudendoclonium, Chlorella, and Chlamydomonas cpDNAs have each lost many genes that are present in Nephroselmis cpDNA, with 18 missing genes shared between all three chlorophyte genomes (table 2). The three chlorophyte genomes share nine rearrangement break points within seven gene clusters that are common to Mesostigma and Nephroselmis (fig. 5), and they all possess SDRs (this study; Maul et al. 2002). In this context, it is interesting to note that intramolecular recombination events between SDRs have been proposed to cause the fragmentation of ancestral operons (Palmer 1991). Altogether, these observations support the notion that the chloroplast genome of the last common ancestor of UTC algae was relatively gene-poor, punctuated with SDRs, and endowed with broken ancestral clusters. Although multiple introns are present in Pseudendoclonium, Chlorella, and Chlamydomonas cpDNAs, there is no evidence that some of these introns took their origin just before the emergence of the UTC clade. Considering that five of the six introns that Pseudendoclonium shares with other green plants are structurally and positionally homologous to chloroplast introns that have been documented only in chlorophycean green algae (table 5), the origin of these introns might be attributed to vertical inheritance from the last common ancestor of ulvophytes and chlorophycean algae. However, given that all five introns, except one (Pa.psbC.1), encode homing endonucleases (table 4) and are thus most probably mobile, we cannot exclude the possibility that horizontal transfer accounts for their presence in both ulvophytes and chlorophycean green algae. Pa.rrl.1, the remaining intron that Pseudendoclonium shares with other green plants, encodes a LAGLIDADG homing endonuclease; interestingly, of all the introns identified so far in green plant cpDNAs, this intron shows the broadest phylogenetic distribution, being found in representatives of the four chlorophyte classes as well as in streptophytes (Lucas et al. 2001; Turmel et al. 2002).
Pseudendoclonium cpDNA features an intermediary level of ancestral characters relative to its Chlorella and Chlamydomonas counterparts. Its gene complement (105 genes) displays more genes than Chlamydomonas cpDNA (94 genes), but fewer than Chlorella cpDNA (112 genes) (table 2). Moreover, ancestral gene clusters have been less extensively rearranged in Pseudendoclonium than in Chlamydomonas, but more in Pseudendoclonium than in Chlorella cpDNAs (fig. 5). Assuming that ancestral characters were lost gradually during evolution, these data would support the idea that the Trebouxiophyceae appeared before the emergence of the Ulvophyceae and Chlorophyceae. However, as loss of ancestral characters is known to occur at variable rates, independent evidence is necessary to validate this hypothesis.
Phylogenetic Inferences from Sequences and Structural Features of the Chloroplast Genome Favor the Hypothesis that the Ulvophyceae Is Sister to the Chlorophyceae
Although our phylogenetic analyses of chloroplast protein and gene sequences have failed to identify unambiguously the divergence order of the UTC lineages, they reject with high confidence the hypothesis that the Ulvophyceae occupies a basal position relative to the Trebouxiophyceae and Chlorophyceae (T3 in fig. 7). About 20 years ago, Mattox and Stewart (1984) proposed this hypothesis based on comparisons of flagellar structure, characteristics of cell division, and nature of cell covering. Our phylogenetic analyses of 58 chloroplast proteins and genes rather favor the idea that the Chlorophyceae diverged first (T1 in fig. 7), but they cannot eliminate the possibility that the Ulvophyceae is sister to the Chlorophyceae (T2 in fig. 7).
Of the latter two hypotheses, we strongly favor that identifying the Ulvophyceae and Chlorophyceae as sister groups because it is robustly supported by three independent sets of data: (1) phylogenetic analysis of gene order data (fig. 8), (2) structural evidence based on derived characters such as shared gene losses and rearrangement break points within ancestrally conserved gene clusters (fig. 7), and (3) phylogenetic analyses of multiple mtDNA-encoded proteins (Pombert et al. 2004). Among the derived cpDNA characters supporting the notion that the Ulvophyceae and Chlorophyceae diverged after the Trebouxiophyceae, we find not only gene losses and rearrangement break points specifically shared between Pseudendoclonium and Chlamydomonas but also shared patterns of expansion for the rpo genes (table 3) and closely related group I introns occupying identical positions. In this context, the occurrence of rpoB and rpoC1 as two distinct ORFs in Chlamydomonas but as single ORFs in Pseudendoclonium suggests that the capture of new sequences by the coding regions of these genes preceded their fragmentation.
Published phylogenies of chlorophytes inferred from nuclear genes (Friedl and O'Kelly 2002) are not in disagreement with the two hypotheses supported by our phylogenetic analyses of cpDNA-encoded sequences. Because interclass relationships are poorly resolved in these nuclear phylogenies based mostly on a single gene (18S rRNA gene), the branching order of the UTC lineages still remains ambiguous in spite of a broad representation of taxa (Friedl and O'Kelly 2002). Although the sequences of green algal organelle genomes offers great potential toward the resolution of interclass relationships, the current data set of whole-genome sequences suffers from very limited taxon sampling. Clearly, the chloroplast and mitochondrial genome sequences of additional chlorophytes will be required to provide unambiguous support for a sister-group relationship between the Ulvophyceae and Chlorophyceae.
Repeated Elements as an Evolutionary Force
From the four chlorophyte chloroplast genome sequences that are currently available, we find that there exists a correlation between the abundance of SDRs and the extent of gene rearrangements. The chloroplast genome of the prasinophyte Nephroselmis contains virtually no SDRs and displays the most ancestral gene organization among the chlorophytes (figs. 5 and 9). Although the cpDNAs of the UTC algae feature SDRs, the abundance of these elements is variable (fig. 9). Chlamydomonas cpDNA displays the greatest density of SDRs and is the most scrambled chlorophyte genome in gene order, whereas Chlorella cpDNA exhibits the lowest density of SDRs and is the least rearranged. Again, Pseudendoclonium cpDNA occupies an intermediate position among the UTC algae with respect to the density of SDRs and the extent of gene rearrangements. The proliferation of SDRs in the chloroplast genome thus appears to be a major cause of genome reorganization in the UTC clade. More chloroplast genome sequences from representatives of each chlorophyte class will be needed to understand the dynamics of SDR evolution.
FIG. 9.— Densities of repeated sequences in chlorophyte cpDNAs as revealed by REPuter. (A) Repeats of 30 nt or more. (B) Repeats of 45 nt or more. For these analyses, one copy of the IR sequence was deleted from the Nephroselmis, Pseudendoclonium, and Chlamydomonas genomes. Repeated elements with identical sequences are connected by lines.
SDRs in green plant cpDNAs could serve as hot spots for nonhomologous recombinational events and lead to inversions when they are in an inverted orientation (Palmer 1991). As indicated by comparative analyses of cpDNAs from numerous land plants (Palmer 1991) and from closely related pairs of chlamydomonads (Boudreau and Turmel 1995, 1996), inversion is most likely the predominant mode of chloroplast gene reorganization. However, for the highly rearranged cpDNAs of the angiosperm family Campanulaceae (Cosner, Raubeson, and Jansen 2004) and subclover (Milligan, Hampton, and Palmer 1989), SDRs have also been proposed to promote transposition events. No sequence elements with characteristics of transposons have been identified in Pseudendoclonium cpDNA; the Wendy element harbored by Chlamydomonas is the only transposon-like sequence that has been found among green plant cpDNAs (Fan, Woelfle, and Mosig 1995).
Supplementary Material
The Pseudendoclonium cpDNA and 18S rDNA sequences reported in this study have been deposited in the GenBank database (accession numbers AY835431 and DQ011230, respectively). All data sets used in phylogenetic analyses are available as supplementary data files. Supplementary Figures S1 and S2 and Supplementary Table S1 are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
Acknowledgements
We are grateful to Patrick Charlebois for his help with the analysis of conserved gene clusters and gene order data. We also thank Charles F. Delwiche and the two anonymous reviewers for their valuable comments and suggestions. 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
One major lineage of green plants, the Chlorophyta, is represented by the green algal classes Prasinophyceae, Ulvophyceae, Trebouxiophyceae, and Chlorophyceae. The Prasinophyceae occupies the most basal position in the Chlorophyta, but the branching order of the Ulvophyceae, Trebouxiophyceae, and Chlorophyceae remains unresolved. The chloroplast genome sequences currently available for representatives of three chlorophyte classes have revealed that this genome is highly plastic, with Chlamydomonas (Chlorophyceae) and Chlorella (Trebouxiophyceae) showing fewer ancestral features than Nephroselmis (Prasinophyceae). We report the 195,867-bp chloroplast DNA (cpDNA) sequence of Pseudendoclonium akinetum (Ulvophyceae), a member of the class that has not been previously examined for detailed cpDNA analysis. This genome shares common evolutionary trends with its Chlorella and Chlamydomonas homologs. The gene content, number of ancestral gene clusters, and abundance of short dispersed repeats in Pseudendoclonium cpDNA are intermediate between those observed for Chlorella and Chlamydomonas cpDNAs. Although Pseudendoclonium cpDNA features a large inverted repeat, its quadripartite structure is unusual in displaying an rRNA operon transcribed toward the large single-copy (LSC) region and a small single-copy region containing 14 genes that are normally found in the LSC region. Twenty-seven group I introns lie in nine genes and fall within four subgroups (IA1, IA2, IA3, and IB); 19 encode putative homing endonucleases, and 7 have homologs at identical insertion sites in other chlorophyte or streptophyte organelle genomes. The high similarity observed among the 14 IA1 and 7 IA2 introns and their encoded endonucleases suggests that many introns arose from intragenomic proliferation of a few founding introns in the lineage leading to Pseudendoclonium. Interestingly, one intron (in atpA) and some of the dispersed repeats also reside in Pseudendoclonium mitochondria, providing strong evidence for interorganellar lateral transfer of these genetic elements. Phylogenetic analyses of 58 cpDNA-encoded proteins and genes support the hypothesis that the Ulvophyceae is sister to the Trebouxiophyceae but cannot eliminate the hypothesis that the Ulvophyceae is sister to the Chlorophyceae. We favor the latter hypothesis because it is strongly supported by phylogenetic analyses of gene order data and by independent structural evidence based on shared gene losses and rearrangement break points within ancestrally conserved gene clusters.
Key Words: green algae ? Ulvophyceae ? Pseudendoclonium akinetum ? chloroplast genome evolution ? group I introns ? repeated sequences
Introduction
The green algae are divided into the phyla Streptophyta and Chlorophyta. The Streptophyta (Bremer 1985) contains all land plants and the green algae belonging to the class Charophyceae (Graham, Cook, and Busse 2000), whereas the Chlorophyta (Sluiman 1985) contains virtually all of the other green algae, i.e., the members of the classes Prasinophyceae, Ulvophyceae, Trebouxiophyceae, and Chlorophyceae (Lewis and McCourt 2004). The basal position of the Prasinophyceae in the Chlorophyta is generally well established, but the branching order of the Ulvophyceae, Trebouxiophyceae, and Chlorophyceae (UTC) remains unresolved (Friedl and O'Kelly 2002; Pombert et al. 2004). A third lineage at the base of the Streptophyta and Chlorophyta is possibly represented by Mesostigma viride; however, some studies suggest that this green alga traditionally classified within the Prasinophyceae represents a basal divergence within the Streptophyta (Bhattacharya et al. 1998; Marin and Melkonian 1999; Karol et al. 2001).
To improve our understanding of phylogenetic relationships among green algae and also to better understand how the chloroplast genome has evolved in this algal group, we have undertaken the complete sequencing of chloroplast DNA (cpDNA) from representatives of various lineages in the Streptophyta and Chlorophyta. Five green algal chloroplast genome sequences are currently available in public databases. The genomes of the prasinophyte Mesostigma (Lemieux, Otis, and Turmel 2000) and of the charophyte Chaetosphaeridium globosum (Turmel, Otis, and Lemieux 2002) most closely resemble their land plant counterparts in terms of overall structure and gene organization. Like most land plant cpDNAs, they feature a quadripartite structure that is characterized by the presence of two copies of an rRNA-encoding inverted repeat (IR) sequence separating a small single-copy (SSC) and a large single-copy (LSC) region. In the Streptophyta, each of these genomic regions shows a highly conserved gene content, and the rRNA operon within the IR is transcribed toward the SSC region.
In contrast, the complete cpDNA sequences of the prasinophyte Nephroselmis olivacea (Turmel, Otis, and Lemieux 1999), the trebouxiophyte Chlorella vulgaris (Wakasugi et al. 1997), and the chlorophycean alga Chlamydomonas reinhardtii (Maul et al. 2002) indicate that the architecture of the chloroplast genome is very fluid in the Chlorophyta. Nephroselmis cpDNA displays ancestral gene clusters and the typical quadripartite structure observed in the streptophytes, whereas Chlorella cpDNA lacks the IR as well as several genes and has gained three group I introns. Chlamydomonas cpDNA is even more scrambled in its structure and gene organization. Although it features an IR, the two single-copy regions are about equal in size and each includes genes usually present in the SSC and LSC regions. Chlamydomonas cpDNA carries fewer genes but more introns than its Chlorella homolog; moreover, it harbors fragmented ancestral operons, and the coding regions of some genes are greatly expanded or broken relative to the corresponding genes in other completely sequenced cpDNAs. Many repeated sequence elements (Maul et al. 2002) are dispersed throughout both the Chlamydomonas and Chlorella genomes; however, such repeats have not been identified in Nephroselmis, Mesostigma, and Chaetosphaeridium cpDNAs.
Here we report the chloroplast genome sequence of Pseudendoclonium akinetum, a unicellular green alga thought to belong to a deep-branching lineage within the Ulvophyceae (Floyd and O'Kelly 1990). This genome has a unique architecture, although it shares some features with Chlorella cpDNA and others with Chlamydomonas cpDNA. Based on our phylogenetic inferences from chloroplast sequences and gene order data as well as on independent structural evidence, we strongly favor the hypothesis that the Ulvophyceae and Chlorophyceae are sister groups.
Materials and Methods
DNA Isolation and Sequencing
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. An A+T–rich fraction containing cpDNA and mitochondrial DNA (mtDNA) was isolated and sequenced as described previously (Pombert et al. 2004). The nuclear-encoded 18S rRNA gene was amplified by polymerase chain reaction (PCR) from total cellular DNA using primers NS1 (White et al. 1990) and 18L (Hamby et al. 1988) as described in Helms et al. (2001). Sequences were edited and assembled with SEQUENCHER 4.2.1 (Gene Codes, Ann Arbor, Mich.).
Sequence Analyses
Genes were identified by Blast homology searches (Altschul et al. 1990) against the nonredundant database of the National Center for Biotechnology Information (NCBI) server (http://www.ncbi.nlm.nih.gov/BLAST/). Positions of open reading frames (ORFs) and protein-coding genes were determined using ORFFINDER at NCBI and various programs of the GCG Wisconsin package (version 10.2) (Accelrys, Burlington, Mass.), whereas genes coding for tRNAs were localized with tRNAscan-SE 1.23 (Lowe and Eddy 1997). Patterns of codon usage for protein-coding genes and ORFs were compared using the CORRESPOND and CODONPREFERENCE programs of the GCG package and the CAI program of the EMBOSS package (version 2.6.0) (http://emboss.sourceforge.net). Repeated sequences were identified with PipMaker (Schwartz et al. 2000) and REPuter 2.74 (Kurtz et al. 2001). Repeats were sorted with REPEATFINDER (Volfovsky, Haas, and Salzberg 2001), and the retrieved classification was refined manually. Putative stem-loop structures and degenerated repeats were identified with PALINDROME and ETANDEM, respectively; these two programs are part of the EMBOSS package. Homologous introns were identified by BlastN searches (Altschul et al. 1990) against the nonredundant database of NCBI using an E value threshold of 1 x 10–6. Homologous introns inserted at identical positions within the same gene were identified by manual screening of the GOBASE database (O'Brien et al. 2003).
Phylogenetic Analyses
The 18S rDNA sequence alignment of Friedl and O'Kelly (2002) was retrieved from TreeBASE (http://www.treebase.org/treebase/), modified to include the P. akinetum sequence, and analyzed as described by these authors. To obtain the chloroplast amino acid data set, sequences of individual chloroplast proteins were aligned using T-COFFEE 1.37 (Notredame, Higgins, and Heringa 2000), the ambiguously aligned regions of these alignments were removed with GBLOCKS 0.91b (Castresana 2000), and the filtered sequences were concatenated. The nucleotide data set was generated by aligning the sequences of individual protein-coding genes on the basis of corresponding protein alignments, filtering the alignments with GBLOCKS 0.91b, concatenating the filtered sequences, and subsequently removing third codon positions with PAUP* 4.0b10 (Swofford 2002). Maximum likelihood (ML) trees inferred from amino acid sequences were computed with PHYML 2.4.4 (Guindon and Gascuel 2003) under the cpREV45 + model (Adachi et al. 2000), whereas ML trees inferred from nucleotide sequences were computed with PAUP* 4.0b10 (Swofford 2002) under the general time reversible + + I model. Modeltest 3.6 (Posada and Crandall 1998) identified the latter model as the one best fitting our nucleotide data. Bootstrap support for each node was calculated using 100 replicates. The confidence limits of alternative tree topologies were evaluated using the Shimodaira-Hasegawa test as implemented in CODEML 3.14 (Yang 1997) and PAUP* 4.0b10 (Swofford 2002). Support for the T1, T2, and T3 topologies by individual proteins in the amino acid data set was estimated with CODEML 3.14 using the MGENE = 1 option.
Intron sequences were aligned manually on the basis of secondary structure predictions, and regions judged to be ambiguously aligned were removed. Neighbor-Joining analyses of the resulting data sets were carried out with PAUP* 4.0b10 (Swofford 2002) using Hasegawa-Kishino-Yano 85 distances with 1,000 bootstrap replicates.
Phylogenetic reconstructions from gene order data were performed with GRAPPA 2.0 (Moret et al. 2001) using the default parameters and the –m (tighter circular lower bound) option. Because this program requires that all genes analyzed be shared between the taxa examined, the genes present within copy B of the IR were excluded from the data set to accommodate the lack of the IR in Chlorella cpDNA.
Results
Phylogenetic Affiliation of P. akinetum
Because it has not been previously demonstrated that P. akinetum (UTEX 1912) belongs to the Ulvophyceae, we have ascertained this affiliation by conducting phylogenetic analyses of nuclear-encoded 18S rDNA sequences with ML, maximum parsimony, and distance methods (Supplementary Fig. S1, Supplementary Material online). Pseudendoclonium akinetum was found to clearly affiliate with other members of the Ulvophyceae, being part of a clade including Pseudendoclonium basiliense and five other members of the Ulotrichales. The P. akinetum and P. basiliense 18S rDNA sequences differ at only seven positions in the data set analyzed.
Genome Structure and Gene Partitioning
Pseudendoclonium cpDNA (195,867 bp) contains two copies of an IR sequence that are separated from one another by LSC and SSC regions (table 1 and fig. 1). The Pseudendoclonium IR encodes only the rRNA operon, in contrast to the IRs of all previously sequenced green plant cpDNAs that include additional genes. Surprisingly, the Pseudendoclonium rRNA operon is transcribed toward the LSC region rather than toward the SSC region. Another unusual feature of Pseudendoclonium cpDNA concerns the pattern of gene partitioning. The SSC region of this genome features 14 genes that map to the LSC region in Mesostigma, Nephroselmis, and land plant cpDNAs (fig. 1). However, the Pseudendoclonium LSC region contains no genes that are usually found in the SSC region.
Table 1 Compared Features of Pseudendoclonium and Other Green Algal cpDNAs
FIG. 1.— Gene map of Pseudendoclonium cpDNA. Genes outside the map are transcribed clockwise. The transcription direction of the rRNA operons is indicated by arrows. The genes shown in yellow, blue, and red map to the IR, LSC, and SSC regions in Mesostigma cpDNA, respectively. Genes and ORFs absent from Mesostigma cpDNA are shown in gray. Gene clusters shared specifically with Chlorella are denoted by thick brackets, whereas the clusters shared with Chlorella and one or more additional chlorophytes are represented by thin brackets. Only the free-standing ORFs larger than 100 codons are shown. tRNA genes are indicated by the one-letter amino acid code followed by the anticodon in parentheses (Me, elongator methionine; Mf, initiator methionine).
Gene Content and Gene Density
Table 2 compares the gene content of Pseudendoclonium cpDNA with those of other green algal cpDNAs. It can be seen that a large number of genes found in both Mesostigma and Nephroselmis (including all ndh genes) have been lost before the emergence of the UTC lineages. With its 105 genes, the chloroplast gene repertoire of Pseudendoclonium is intermediate in size between those of Chlorella and Chlamydomonas (see table 1). Eight of the genes identified in Chlorella (chlB, chlL, chlN, cysA, cysT, trnL(gag), trnS(gga), and trnT(ggu)) are absent from Pseudendoclonium, whereas all of the genes found in Pseudendoclonium except trnR(ccu) are present in Chlorella. Fourteen of the genes identified in Pseudendoclonium (accD, chlI, infA, minD, psaI, psaM, rpl12, rpl19, rpl32, ycf20, ycf62, trnL(caa), trnR(ccg), and trnR(ccu)) are absent from Chlamydomonas, whereas all of the genes found in Chlamydomonas except chlB, chlL, and chlN are present in Pseudendoclonium.
Table 2 Gene Content in Pseudendoclonium and Other Green Algal cpDNAs
A total of 19 of the 27 introns displayed by Pseudendoclonium cpDNA carry an internal ORF encoding a putative homing endonuclease (see table 4). In addition to these intron ORFs, 12 free-standing ORFs larger than 100 codons are present in Pseudendoclonium cpDNA; only 2 (orf286 and orf521) show a codon usage not significantly different from that observed for the protein-coding genes.
Table 4 Group I Introns in Pseudendoclonium cpDNA
Genes are less tightly packed in Pseudendoclonium cpDNA than in Mesostigma and Nephroselmis cpDNAs (table 1). The intergenic spacers in this ulvophyte genome are up to 3,215 bp in size, with an average of 600 bp. The higher proportion of coding sequences found in Pseudendoclonium cpDNA relative to Chlorella cpDNA is explained by the greater number of introns in the former genome (table 1).
Expansion of Coding Regions
Eight protein-coding genes in Pseudendoclonium cpDNA are nearly two or three times larger than their Mesostigma homologs (table 3). For all of these genes, with the exception of cemA, expansion of coding regions appears to be the main cause of their increased sizes. Because the sizes of the corresponding Mesostigma genes match those of their counterparts in streptophytes and the cyanobacterium Synechocystis sp. PCC 6803, we eliminated the alternative hypothesis that shrinkage of the Mesostigma coding regions is responsible for the observed size variations. The ftsH, rpoB, rpoC1, and rpoC2 genes represent the most notable cases of gene expansion; the levels of expansion noted for these genes are comparable or greater than those for the corresponding genes in Chlamydomonas (table 3). The extra sequences accounting for the expansion of the Pseudendoclonium ftsH and rpo genes map mainly to internal coding regions. In Chlamydomonas, rpoB, rpoC1, as well as rps2 each consist of two separate ORFs, and even though these ORFs are adjacent, no intron has been detected in the noncoding sequence separating them. In Pseudendoclonium, each of these three genes occurs as a single ORF.
Table 3 Compared Sizes of Expanded Genes in Pseudendoclonium, Chlorella, and Chlamydomonas cpDNAs
Introns
The 27 introns in Pseudendoclonium cpDNA account for 14.8% of the genome size and are preferentially located in genes coding for photosynthetic proteins (table 4). They all belong to the group I family and fall within subgroups IA1, IA2, IA3, and IB according to the classification system of Michel and Westhof (1990). The subgroup IA1 is well represented by the Pseudendoclonium introns, with 14 introns assigned to this category.
Structural and phylogenetic analyses of the IA1 and IA2 introns revealed close relationships among members of the same subgroup. These introns are well conserved in both primary sequences and secondary structures (fig. 2A, B, C, and D); moreover, those sharing similar ORFs tend to cluster together in phylogenetic trees inferred from core sequences (compare fig. 2E with fig. 2C and D). The seven IA1 intron-encoded H-N-H endonucleases are all specified by an ORF in L5 (table 4) and can be assigned to three categories based on sequence similarity within the region containing the H-N-H motif (fig. 2E). Pa.psbA.1 differs from the other IA1 introns in carrying an ORF in L9 that potentially codes for an endonuclease of the LAGLIDADG family (fig. 2E). Five IA2 introns exhibit an ORF in L6 that codes for a putative GIY-YIG endonuclease; these IA2 intron-encoded proteins can be divided into two categories based on sequence similarity (fig. 2D and E).
FIG. 2.— Comparative analyses of group I introns and intron ORFs in Pseudendoclonium cpDNA. (A and B) Consensus secondary structure models of introns displayed according to Burke et al. (1987): (A) IA1 introns and (B) IA2 introns. Arrows denote the 5' splice sites between exon and intron residues. Highly conserved residues (in 11 IA1 and 5 IA2 introns) and slightly less conserved residues (in nine IA1 and four IA2 introns) are shown in uppercase and lowercase characters, respectively; the other residues are represented by filled circles. Conserved base pairings (in 11 IA1 and 6 IA2 introns) are denoted by bars. Numbers inside the variable loops indicate the size variations of these loops in the compared introns. Note that the P2 pairing is missing in one IA1 intron (Pa.psbA.7). (C and D) Neighbor-Joining analyses of intron core sequences: (C) IA1 introns and (D) IA2/IA3 introns. The Chlamydomonas reinhardtii (Cr) and Chlamydomonas eugametos (Ce) introns homologous to the Pseudendoclonium (Pa) introns (see table 5) were included in these analyses. Nodes that were identified in at least 80% of the bootstrap replicates are labeled with asterisks. The introns denoted in green, red, blue, and orange encode putative homing endonucleases; in each panel, those denoted by the same color specify closely related proteins. (E) Alignment of the endonuclease regions containing the H-N-H, LAGLIDADG, or GIY-YIG motif. The amino acid residues making up each motif are underlined.
Table 5 Group I Introns at Identical Gene Locations in Pseudendoclonium cpDNA, Other Green Algal cpDNAs, and Land Plant cpDNAs
The IA3 and IB introns show more sequence divergence than their IA1 and IA2 counterparts. As shown in Supplementary Figure S2 (Supplementary Material online) and table 4, the two IA3 introns (Pa.psbA.3 and Pa.rrl.1) differ greatly with respect to their core structures and encoded endonucleases. The P3.1 and P3.2 pairings are present in Pa.psbA.3 but not in Pa.rrl.1, and Pa.psbA.3 encodes a putative GIY-YIG endonuclease, whereas Pa.rrl.1 encodes a putative endonuclease with a single LAGLIDADG motif (table 4). Each of the four IB introns contains an ORF in L8 that codes for a putative LAGLIDADG endonuclease with one or two copies of this motif (table 4); however, these ORFs are distantly related in sequence.
Our BlastN searches of the GenBank database for homologs of the Pseudendoclonium chloroplast introns identified only introns from green algae and land plants (E value threshold of 1x 10–6). The most surprising result of this analysis was the finding of homologous introns (Pa.atpA.1 and Pa.atp1.1) inserted at the same gene position in the chloroplast and mitochondrial genomes of Pseudendoclonium (fig. 3). In addition to highly similar primary sequences and secondary structures, these introns feature in L8 very similar ORFs encoding putative endonucleases with a double LAGLIDADG motif.
FIG. 3.— Comparative analyses of the chloroplast Pa.atpA.1 and mitochondrial Pa.atp1.1 introns. (A) Consensus secondary structure models of the two introns displayed according to Burke et al. (1987). The arrow denotes the 5' splice site between the exon and intron residues. Identical residues are shown in uppercase characters, whereas different residues are represented by filled circles. Conserved base pairings are denoted by bars. Upper and lower numbers inside the variable loops indicate size variations in the Pa.atpA.1 and Pa.atp1.1 introns, respectively. (B) ClustalW alignment of the putative LAGLIDADG endonucleases encoded by the two introns. The LAGLIDADG motifs are underlined.
Table 5 reports the homologous group I introns of chloroplast origin that proved to be inserted at the same sites as their Pseudendoclonium counterparts. Five Pseudendoclonium introns have known homologs in chlorophycean green algae, whereas homologs of Pa.rrl.1 have been observed in prasinophytes, trebouxiophytes, chlorophycean green algae, and the hornwort Anthoceros punctatus. The three psbA and rrl introns in C. reinhardtii cpDNA exhibit a high degree of primary sequence and secondary structure conservation with their Pseudendoclonium counterparts (Supplementary Fig. S2, Supplementary Material online). Even the unusual P3.1 and P3.2 pairings found in Pa.psbA.3 are present in Cr.psbA.2.
It is intriguing that L9 of Pa.psbA.1 codes for a putative single LAGLIDADG endonuclease and that no endonuclease motif has been assigned to the ORF of 102 codons found within the same loop in the homologous Cr.psbA.1 IA1 intron (Holloway, Deshpande, and Herrin 1999). Our analysis of the published Chlamydomonas cpDNA sequence mapping at this locus revealed the presence of an ORF of 46 codons encoding a LAGLIDADG motif 29 nt upstream of orf102; interestingly, the predicted protein sequence of this ORF exhibits high similarity with the N-terminal domain of the Pa.psbA.1-encoded endonuclease (fig. 2E). To test the possibility that there is a frameshift in the sequence originally published by Holloway, Deshpande, and Herrin (1999), we sequenced a PCR product containing the Cr.psbA.1 intron; however, we found no nucleotide difference. We conclude that a nonsense mutation led to loss of the LAGLIDADG motif in the Cr.psbA.1-encoded protein.
Repeated Elements
The Pseudendoclonium chloroplast genome contains a large number of repeated elements (fig. 4). Two types of repeated sequences can be distinguished: short tandem repeats and short dispersed repeats (SDRs). The short tandem repeats are found in the vicinity of trnMe(cau) in a region spanning about 2 kb. The units forming this repeat region are 10–20 bp in size, rich in A + T, and degenerated in sequence.
FIG. 4.— PipMaker analysis of Pseudendoclonium cpDNA. This genome sequence was aligned against itself. At the top of the alignment, genes and their polarities are denoted by horizontal arrows, and coding sequences are represented by filled boxes. The SDR repeat units described in table 6 are denoted by boxed letters. The short tandem repeats near trnMe(cau) are shown as gray dots. Similarities between aligned regions are shown as average percent identity between 50% and 100% identity.
Table 6 SDR Repeat Units in Pseudendoclonium cpDNA and mtDNA
The SDRs map to intergenic spacers and/or introns and can be classified into four groups of repeat units (A, B, C, and D) on the basis of their primary sequences (table 6). They occur frequently as palindromic sequences separated by 7–8 bp, and the stem-loop structures often contain more than one SDR unit. As deletion of palindromic structures and other repeated elements is known to occur during cloning in Escherichia coli, we confirmed by direct sequencing of PCR products the sizes and sequences of the Pseudendoclonium SDRs that we identified by analysis of cloned fragments. To our surprise, we found that numerous SDRs in Pseudendoclonium cpDNA bear identical or close sequence similarity to those present in the mitochondrial genome of this alga (Pombert et al. 2004). These homologous mitochondrial SDRs fall within three of the four above-mentioned groups of repeat units (A, B, and C) (table 6) and also occur as stem-loop structures composed of one or more repeat units. To our knowledge, the presence of closely related SDRs in different organelles of the same eukaryotic cell has not been previously reported.
Genome Organization
Our pairwise comparisons of Pseudendoclonium cpDNA with previously sequenced green plant cpDNAs indicate that its gene organization is most similar to that of Chlorella. These two green algal genomes share 16 distinct gene clusters, 3 of which (rps18-rpl20, psbK-ycf12-psaM, and trnE(uuc)-trnMf(cau)) have not been identified in any other sequenced chloroplast genomes (fig. 1). A single gene cluster (trnC(gca)-trnT(ugu)) is shared specifically between Pseudendoclonium and Chlamydomonas cpDNAs.
Twelve of the gene clusters that are conserved between Mesostigma and Nephroselmis cpDNAs have been broken in Pseudendoclonium cpDNA (fig. 5). As revealed by our analysis of the rearrangement break points within these ancestrally conserved clusters, the Pseudendoclonium genome shares 9 of the 10 break points found in Chlorella cpDNA and features 13 additional break points (fig. 5). All of these nine break points are also observed in Chlamydomonas cpDNA (fig. 5). Of the 33 break points exhibited by this chlamydomonad genome, 11 are specifically shared with Pseudendoclonium, whereas a single one is specifically shared with Chlorella. The psbB-psbT-psbN-psbH cluster displays only two break points that are unique to Pseudendoclonium. Although the psbN gene of this ulvophyte has retained its location between psbT and psbH, it has been relocated to the DNA strand encoding the other psb genes (fig. 1).
FIG. 5.— Fragmented ancestral gene clusters in Pseudendoclonium, Chlorella, and Chlamydomonas cpDNAs. The indicated clusters are found in both Mesostigma and Nephroselmis cpDNAs. Note that rpl22 has not been represented in the large ribosomal protein gene cluster; this gene is found in Mesostigma between rps19 and rps3 but is missing from Nephroselmis and the three other chlorophytes. Sites of fragmentation are denoted by arrowheads above the clusters, with the arrowheads at the lower, middle, and upper positions pointing to sites in Chlamydomonas, Pseudendoclonium, and Chlorella, respectively. Genes missing from Chlamydomonas, Pseudendoclonium, and Chlorella are denoted by circles, asterisks, and double dagger, respectively, below the clusters. Gene polarities are not shown.
Phylogenetic Analyses
The amino acid and nucleotide sequences derived from the 58 protein-coding genes (see Supplementary Table S1, Supplementary Material online) that are shared between the cpDNAs of Pseudendoclonium, Chaetosphaeridium, Chlamydomonas, Chlorella, Marchantia, Nephroselmis, and Nicotiana were concatenated and analyzed with ML inference methods using the homologous sequences of Mesostigma as the outgroup. As expected, analyses of both the amino acid (11,225 positions) and nucleotide (25,318 positions) data sets yielded a best tree (T1) in which the chlorophytes and streptophytes form two distinct lineages (fig. 6). In the chlorophyte lineage, the prasinophyte Nephroselmis occupies the most basal position, whereas the trebouxiophyte Chlorella, the ulvophyte Pseudendoclonium, and chlorophycean alga Chlamydomonas form a clade in which Pseudendoclonium is sister to Chlorella. Topology T1 accounted for 91% and 80% of the bootstrap replicates in the analyses of the amino acid and nucleotide data sets, respectively, and was also found to be the most highly supported topology in distance (ML and LogDet distances) and maximum parsimony analyses (data not shown). In all analyses, instability in the branching order of taxa was observed only for the UTC clade. The alternative topology showing Pseudendoclonium as sister to Chlamydomonas (T2) was recovered in 9% and 20% of the bootstrap replicates in the ML analyses of the amino acid and nucleotide data sets, respectively, whereas the topology placing Pseudendoclonium at a basal position in the UTC clade (T3) was not detected in any of the 100 bootstrap replicates. The confidence levels of these alternative topologies were assessed using the statistical test of Shimodaira-Hasegawa. T3, but not T2, proved to be significantly worse than T1 at P < 0.05 in the analyses of both the amino acid and nucleotide data sets (fig. 7).
FIG. 6.— Phylogenetic position of Pseudendoclonium as inferred by ML analyses of 58 cpDNA-encoded proteins and genes. (A) Best protein tree. (B) Best gene tree. Bootstrap values are indicated on the corresponding nodes, and branch lengths are drawn to scale.
FIG. 7.— Evaluation of the three phylogenetic hypotheses for the branching order of UTC taxa using the Shimodaira-Hasegawa test and structural evidence based on shared gene losses and rearrangement break points within ancestrally conserved gene clusters. Values above and below the nodes indicate the number of break points and gene losses, respectively. The P values obtained in the Shimodaira-Hasegawa test are indicated for the ML analyses of both the amino acid (aa) and nucleotide (nt) data sets.
Separate ML analyses of the amino acid data set with CODEML revealed no strong disagreement among the phylogenetic signals provided by the individual proteins (Supplementary Table S1, Supplementary Material online). Topologies T1, T2, and T3 were found to be supported by 22, 25, and 11 proteins, respectively, with RELL bootstrap values ranging from 39% to 93%. However, all 58 proteins, with the exception of rpoB, failed to provide a signal of sufficient strength to reject one or both of the alternative topologies at P < 0.05 in the Shimodaira-Hasegawa test. rpoB supported T1 and rejected T3 (but not T2) at P = 0.02. Thus, it appears that general homoplasy throughout the data set is the most likely explanation for the lack of resolution between T1 and T2.
Phylogenetic relationships were also inferred from gene order data using the relative positions of the 80 chloroplast genes shared by Pseudendoclonium and the seven green plants mentioned above. GRAPPA 2.0, the program used for these analyses, reconstructs phylogenies by assuming that gene rearrangements occur by inversions. The best tree was found to display the T2 topology and to feature a total length of 240 inversion steps (fig. 8). User-tree analyses constrained to the T1 and T3 topologies yielded trees with 6 and 10 extra steps, respectively.
FIG. 8.— Phylogenetic position of Pseudendoclonium as inferred by gene order analysis of 80 chloroplast genes. The best tree is shown with branch lengths drawn to scale.
Mapping of shared gene losses and rearrangement break points located within ancestrally conserved gene clusters on topologies T1, T2, and T3 also revealed that these two sets of structural characters independently support topology T2 as the most parsimonious scenario (fig. 7). A total of 36 rearrangement break points and 41 gene losses were mapped on T2 compared to 47 break points and 46 gene losses on T1 and 46 break points and 45 gene losses on T3. In contrast to T1 and T3, T2 shows several rearrangement events and gene losses that are specifically shared between sister taxa in the UTC lineage.
Discussion
Unusual Features of Pseudendoclonium cpDNA
Like the chloroplast genomes of the chlorophycean green algae belonging to the genus Chlamydomonas, Pseudendoclonium cpDNA is unusual with respect to its quadripartite structure (fig. 1). Fourteen of the genes found in the SSC region of this ulvophyte genome map to a different genomic region (most often the LSC region) in all streptophytes featuring an IR as well as in the prasinophytes Nephroselmis and Mesostigma. Moreover, the Pseudendoclonium IR features an rRNA operon that is transcribed toward the LSC region. During the evolution of ulvophytes, the transfer of genes from the LSC to the SSC region may have been closely associated with the inversion of the IR. In this context, it may be envisioned that an ancestral genome with a conventional quadripartite structure has undergone two consecutive inversions, each involving a segment encompassing the IR and part of the LSC or, alternatively, one inversion involving a segment encompassing the entire IR sequence and the other the second IR sequence. Whatever the mechanism, additional ulvophyte chloroplast genomes will need to be examined to determine whether the atypical quadripartite structure reported here for Pseudendoclonium cpDNA is commonly found in the Ulvophyceae. Prior to our study, the only ulvophyte chloroplast genome that had been investigated by physical and gene mapping is that of Codium fragile, a green alga thought to belong to a later-diverging lineage than the Pseudendoclonium lineage (Manhart et al. 1989). Considering that Codium cpDNA is highly reduced in size (89 kb), lacks an IR, and has a broken rRNA operon, the chloroplast genome appears to have evolved under relaxed constraints in the Ulvophyceae.
Another unusual feature of Pseudendoclonium cpDNA concerns its intron composition (table 4). Of all the chloroplast genome sequences reported so far, this chlorophyte genome is the richest in group I introns. The striking similarity among the 14 IA1 and 7 IA2 introns and the putative homing endonucleases encoded by these introns (fig. 2) suggests that many of the 27 group I introns found in Pseudendoclonium cpDNA arose from intragenomic proliferation of a few founding introns in the lineage leading to Pseudendoclonium. The possibility that numerous Pseudendoclonium introns took their origin from independent invasions of closely related introns by lateral transfer rather than by intragenomic proliferation seems less likely but cannot be excluded. Interestingly, the intron composition of Pseudendoclonium cpDNA resembles that of its mitochondrial counterpart (Pombert et al. 2004) in featuring exclusively group I introns, a substantial fraction of which specifies potential homing endonucleases.
The highly similar group IB introns sharing the same insertion site in the Pseudendoclonium chloroplast atpA and mitochondrial atp1 genes (fig. 3) as well as the closely related SDRs in both organelle DNAs of this green alga (table 6) provide compelling evidence for interorganellar, lateral transfer of these genetic elements. The lateral transfer of the atpA/atp1 introns appears to have occurred specifically in the ulvophyte lineage, considering that these introns represent the first examples of intron insertion at their cognate site in atpA/atp1. Cases of interorganellar transfers of group I introns have been previously documented for green algae (Turmel, Mercier, and C?té 1993; Turmel et al. 1995, 1999); however, the rDNA introns associated with these transfers exhibit a sporadic distribution pattern and occur in more than one chlorophyte lineage, making it difficult to estimate the timing of the lateral transfer event(s).
The cpDNAs of Pseudendoclonium and Other Advanced Chlorophytes Share Common Evolutionary Trends
A number of shared derived features between the cpDNAs of Pseudendoclonium and other advanced chlorophytes highlight common evolutionary trends among the members of the UTC clade. Like its Pseudendoclonium homolog, the completely sequenced cpDNA of the chlorophycean green alga C. reinhardtii as well as other chlamydomonad cpDNAs that have been analyzed by physical and gene mapping display an atypical quadripartite structure in which gene partitioning among the two single-copy regions differs from the conserved pattern observed in all other green plant cpDNAs containing an IR (Boudreau, Otis, and Turmel 1994; Boudreau and Turmel 1995, 1996; Maul et al. 2002). The single-copy regions of these genomes are about equal in size, and their genes have been so extensively shuffled that it is impossible to trace the events that led to their relocations and to determine if the rRNA operons have been altered in their orientation. On the other hand, studies of C. vulgaris and Chlorella ellipsoidea cpDNAs clearly indicate that the IR was the subject of rearrangement or deletion during the evolution of trebouxiophytes (Yamada 1991; Wakasugi et al. 1997). Given the absence of the IR in C. vulgaris and the fragmentary information currently available for the IR-containing cpDNA of C. ellipsoidea, it remains unknown if genes were relocated from one single-copy region to the other. Unlike its C. vulgaris homolog which is intact, the C. ellipsoidea rRNA operon is fragmented into two pieces, one containing the rrs and trnI(gau) genes and the other containing the trnA(ugc), rrl, and rrf genes (Yamada and Shimaji 1987). The orientation of these genes is such that a single inversion of the latter fragment would recreate an intact rRNA operon that is transcribed toward the LSC region. From these data alone, one cannot conclude about the timing of the event(s) that led to the change in orientation of the Pseudendoclonium and C. ellipsoidea rRNA operons. It is possible that the chloroplast genome of the last common ancestor of the UTC algae had already acquired an IR with an rRNA operon transcribed toward the LSC region and an SSC region with some genes usually present in the opposite single-copy region.
UTC algae also exhibit common features at the levels of gene content, gene order, repeated sequences, and intron content. Pseudendoclonium, Chlorella, and Chlamydomonas cpDNAs have each lost many genes that are present in Nephroselmis cpDNA, with 18 missing genes shared between all three chlorophyte genomes (table 2). The three chlorophyte genomes share nine rearrangement break points within seven gene clusters that are common to Mesostigma and Nephroselmis (fig. 5), and they all possess SDRs (this study; Maul et al. 2002). In this context, it is interesting to note that intramolecular recombination events between SDRs have been proposed to cause the fragmentation of ancestral operons (Palmer 1991). Altogether, these observations support the notion that the chloroplast genome of the last common ancestor of UTC algae was relatively gene-poor, punctuated with SDRs, and endowed with broken ancestral clusters. Although multiple introns are present in Pseudendoclonium, Chlorella, and Chlamydomonas cpDNAs, there is no evidence that some of these introns took their origin just before the emergence of the UTC clade. Considering that five of the six introns that Pseudendoclonium shares with other green plants are structurally and positionally homologous to chloroplast introns that have been documented only in chlorophycean green algae (table 5), the origin of these introns might be attributed to vertical inheritance from the last common ancestor of ulvophytes and chlorophycean algae. However, given that all five introns, except one (Pa.psbC.1), encode homing endonucleases (table 4) and are thus most probably mobile, we cannot exclude the possibility that horizontal transfer accounts for their presence in both ulvophytes and chlorophycean green algae. Pa.rrl.1, the remaining intron that Pseudendoclonium shares with other green plants, encodes a LAGLIDADG homing endonuclease; interestingly, of all the introns identified so far in green plant cpDNAs, this intron shows the broadest phylogenetic distribution, being found in representatives of the four chlorophyte classes as well as in streptophytes (Lucas et al. 2001; Turmel et al. 2002).
Pseudendoclonium cpDNA features an intermediary level of ancestral characters relative to its Chlorella and Chlamydomonas counterparts. Its gene complement (105 genes) displays more genes than Chlamydomonas cpDNA (94 genes), but fewer than Chlorella cpDNA (112 genes) (table 2). Moreover, ancestral gene clusters have been less extensively rearranged in Pseudendoclonium than in Chlamydomonas, but more in Pseudendoclonium than in Chlorella cpDNAs (fig. 5). Assuming that ancestral characters were lost gradually during evolution, these data would support the idea that the Trebouxiophyceae appeared before the emergence of the Ulvophyceae and Chlorophyceae. However, as loss of ancestral characters is known to occur at variable rates, independent evidence is necessary to validate this hypothesis.
Phylogenetic Inferences from Sequences and Structural Features of the Chloroplast Genome Favor the Hypothesis that the Ulvophyceae Is Sister to the Chlorophyceae
Although our phylogenetic analyses of chloroplast protein and gene sequences have failed to identify unambiguously the divergence order of the UTC lineages, they reject with high confidence the hypothesis that the Ulvophyceae occupies a basal position relative to the Trebouxiophyceae and Chlorophyceae (T3 in fig. 7). About 20 years ago, Mattox and Stewart (1984) proposed this hypothesis based on comparisons of flagellar structure, characteristics of cell division, and nature of cell covering. Our phylogenetic analyses of 58 chloroplast proteins and genes rather favor the idea that the Chlorophyceae diverged first (T1 in fig. 7), but they cannot eliminate the possibility that the Ulvophyceae is sister to the Chlorophyceae (T2 in fig. 7).
Of the latter two hypotheses, we strongly favor that identifying the Ulvophyceae and Chlorophyceae as sister groups because it is robustly supported by three independent sets of data: (1) phylogenetic analysis of gene order data (fig. 8), (2) structural evidence based on derived characters such as shared gene losses and rearrangement break points within ancestrally conserved gene clusters (fig. 7), and (3) phylogenetic analyses of multiple mtDNA-encoded proteins (Pombert et al. 2004). Among the derived cpDNA characters supporting the notion that the Ulvophyceae and Chlorophyceae diverged after the Trebouxiophyceae, we find not only gene losses and rearrangement break points specifically shared between Pseudendoclonium and Chlamydomonas but also shared patterns of expansion for the rpo genes (table 3) and closely related group I introns occupying identical positions. In this context, the occurrence of rpoB and rpoC1 as two distinct ORFs in Chlamydomonas but as single ORFs in Pseudendoclonium suggests that the capture of new sequences by the coding regions of these genes preceded their fragmentation.
Published phylogenies of chlorophytes inferred from nuclear genes (Friedl and O'Kelly 2002) are not in disagreement with the two hypotheses supported by our phylogenetic analyses of cpDNA-encoded sequences. Because interclass relationships are poorly resolved in these nuclear phylogenies based mostly on a single gene (18S rRNA gene), the branching order of the UTC lineages still remains ambiguous in spite of a broad representation of taxa (Friedl and O'Kelly 2002). Although the sequences of green algal organelle genomes offers great potential toward the resolution of interclass relationships, the current data set of whole-genome sequences suffers from very limited taxon sampling. Clearly, the chloroplast and mitochondrial genome sequences of additional chlorophytes will be required to provide unambiguous support for a sister-group relationship between the Ulvophyceae and Chlorophyceae.
Repeated Elements as an Evolutionary Force
From the four chlorophyte chloroplast genome sequences that are currently available, we find that there exists a correlation between the abundance of SDRs and the extent of gene rearrangements. The chloroplast genome of the prasinophyte Nephroselmis contains virtually no SDRs and displays the most ancestral gene organization among the chlorophytes (figs. 5 and 9). Although the cpDNAs of the UTC algae feature SDRs, the abundance of these elements is variable (fig. 9). Chlamydomonas cpDNA displays the greatest density of SDRs and is the most scrambled chlorophyte genome in gene order, whereas Chlorella cpDNA exhibits the lowest density of SDRs and is the least rearranged. Again, Pseudendoclonium cpDNA occupies an intermediate position among the UTC algae with respect to the density of SDRs and the extent of gene rearrangements. The proliferation of SDRs in the chloroplast genome thus appears to be a major cause of genome reorganization in the UTC clade. More chloroplast genome sequences from representatives of each chlorophyte class will be needed to understand the dynamics of SDR evolution.
FIG. 9.— Densities of repeated sequences in chlorophyte cpDNAs as revealed by REPuter. (A) Repeats of 30 nt or more. (B) Repeats of 45 nt or more. For these analyses, one copy of the IR sequence was deleted from the Nephroselmis, Pseudendoclonium, and Chlamydomonas genomes. Repeated elements with identical sequences are connected by lines.
SDRs in green plant cpDNAs could serve as hot spots for nonhomologous recombinational events and lead to inversions when they are in an inverted orientation (Palmer 1991). As indicated by comparative analyses of cpDNAs from numerous land plants (Palmer 1991) and from closely related pairs of chlamydomonads (Boudreau and Turmel 1995, 1996), inversion is most likely the predominant mode of chloroplast gene reorganization. However, for the highly rearranged cpDNAs of the angiosperm family Campanulaceae (Cosner, Raubeson, and Jansen 2004) and subclover (Milligan, Hampton, and Palmer 1989), SDRs have also been proposed to promote transposition events. No sequence elements with characteristics of transposons have been identified in Pseudendoclonium cpDNA; the Wendy element harbored by Chlamydomonas is the only transposon-like sequence that has been found among green plant cpDNAs (Fan, Woelfle, and Mosig 1995).
Supplementary Material
The Pseudendoclonium cpDNA and 18S rDNA sequences reported in this study have been deposited in the GenBank database (accession numbers AY835431 and DQ011230, respectively). All data sets used in phylogenetic analyses are available as supplementary data files. Supplementary Figures S1 and S2 and Supplementary Table S1 are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
Acknowledgements
We are grateful to Patrick Charlebois for his help with the analysis of conserved gene clusters and gene order data. We also thank Charles F. Delwiche and the two anonymous reviewers for their valuable comments and suggestions. 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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