The chloroplast genome sequence of the green alga Leptosira terrestris: multiple losses of the inverted repeat and extensive genome rearrangements within the Trebouxiophyceae
© de Cambiaire et al; licensee BioMed Central Ltd. 2007
Received: 21 March 2007
Accepted: 04 July 2007
Published: 04 July 2007
In the Chlorophyta – the green algal phylum comprising the classes Prasinophyceae, Ulvophyceae, Trebouxiophyceae and Chlorophyceae – the chloroplast genome displays a highly variable architecture. While chlorophycean chloroplast DNAs (cpDNAs) deviate considerably from the ancestral pattern described for the prasinophyte Nephroselmis olivacea, the degree of remodelling sustained by the two ulvophyte cpDNAs completely sequenced to date is intermediate relative to those observed for chlorophycean and trebouxiophyte cpDNAs. Chlorella vulgaris (Chlorellales) is currently the only photosynthetic trebouxiophyte whose complete cpDNA sequence has been reported. To gain insights into the evolutionary trends of the chloroplast genome in the Trebouxiophyceae, we sequenced cpDNA from the filamentous alga Leptosira terrestris (Ctenocladales).
The 195,081-bp Leptosira chloroplast genome resembles the 150,613-bp Chlorella genome in lacking a large inverted repeat (IR) but differs greatly in gene order. Six of the conserved genes present in Chlorella cpDNA are missing from the Leptosira gene repertoire. The 106 conserved genes, four introns and 11 free standing open reading frames (ORFs) account for 48.3% of the genome sequence. This is the lowest gene density yet observed among chlorophyte cpDNAs. Contrary to the situation in Chlorella but similar to that in the chlorophycean Scenedesmus obliquus, the gene distribution is highly biased over the two DNA strands in Leptosira. Nine genes, compared to only three in Chlorella, have significantly expanded coding regions relative to their homologues in ancestral-type green algal cpDNAs. As observed in chlorophycean genomes, the rpoB gene is fragmented into two ORFs. Short repeats account for 5.1% of the Leptosira genome sequence and are present mainly in intergenic regions.
Our results highlight the great plasticity of the chloroplast genome in the Trebouxiophyceae and indicate that the IR was lost on at least two separate occasions. The intriguing similarities of the derived features exhibited by Leptosira cpDNA and its chlorophycean counterparts suggest that the same evolutionary forces shaped the IR-lacking chloroplast genomes in these two algal lineages.
All chloroplasts of photosynthetic eukaryotes inherited from their cyanobacterial ancestors a reduced genome that encodes part of the genes essential for their biogenesis. The chloroplast genome has been studied in various algal lineages, particularly in the green algal/land plant lineage (Viridiplantae) for which the number of complete chloroplast DNA (cpDNA) sequences available in public databases increases steadily. Comparative analyses of the latter genome sequences highlight distinct evolutionary trends in the Streptophyta and the Chlorophyta. In the Streptophyta, the division comprising the green algae from the class Charophyceae and all land plants, the chloroplast genome shows remarkable conservation in overall structure, gene content, gene order and intron content [1, 2]. In contrast, considerable fluidity in chloroplast genome organization is the hallmark of the Chlorophyta, the division comprising the four remaining green algal classes (Prasinophyceae, Ulvophyceae, Trebouxiophyceae and Chlorophyceae).
The chloroplast genomes from members of the Ulvophyceae (Oltmannsiellopsis viridis  and Pseudendoclonium akinetum ), Trebouxiophyceae (Chlorella vulgaris  and the non-photosynthetic alga Helicosporidium sp. ) and Chlorophyceae (Stigeoclonium helveticum , Scenedesmus obliquus  and Chlamydomonas reinhardtii ) display various patterns of divergence compared to the ancestral pattern described for a representative of the Prasinophyceae (Nephroselmis olivacea ). The cpDNA of this prasinophyte resembles most of its streptophyte homologues in displaying small and large single-copy (SSC and LSC) regions that are separated from one another by two identical inverted repeat regions (IR). Moreover, the set of genes encoded by each of these three genomic regions is almost identical in Nephroselmis and streptophyte cpDNAs. Moderate deviations from this ancestral-type architecture are seen in the two earliest-diverging lineages of the Ulvophyceae (the orders Oltmannsiellopsidales and Ulotrichales), supporting the hypothesis that a dozen genes were transferred from the LSC to the SSC region and that the rRNA operon in the IR was altered in orientation very early during the evolution of ulvophytes . Although the cpDNAs of the chlorophycean green algae Chlamydomonas and Scenedesmus have also retained a quadripartite structure, the single-copy regions of these genomes differ extensively in gene content and both genomes deviate radically from the ancestral gene partitioning pattern . The chloroplast genomes of Chlorella  and Stigeoclonium  as well as the plastid genome of Helicosporidium  lack an IR, indicating that this repeat was lost independently in the Trebouxiophyceae and Chlorophyceae. Considering that loss of the IR is often correlated with gene rearrangements [11, 12], it is noteworthy that the IR-lacking cpDNA of the trebouxiophyte Chlorella retains an almost intact pattern of ancestral gene partitioning .
In Helicosporidium and the three lineages of chlorophycean green algae examined, remodelling of plastid/chloroplast genome architecture was accompanied by the formation of long blocks of consecutive genes on the same DNA strand [6–8, 13]. For the Stigeoclonium and Helicosporidium genomes, the pattern of gene distribution between the two DNA strands was found to be correlated with a bias in base composition along each strand, allowing the identification of a potential origin of replication [6, 7].
The cpDNAs of ulvophyte, trebouxiophyte and chlorophycean (UTC) green algae also underwent erosion of ancestral gene clusters, many gene losses, proliferation of short dispersed repeats and introns as well as expansions of intergenic spacers and proteins-coding genes [3, 7]. All photosynthetic genes were lost in the lineage leading to the heterotrophic trebouxiophyte Helicosporidium, giving rise to an extremely compact genome greatly reduced in both size and gene content . In addition, three expanded genes in chlorophycean green algal cpDNAs were split into two distinct open reading frames (ORFs); this is the case for the rpoB genes of Stigeoclonium, Scenedesmus and Chlamydomonas, for rps2 of the latter two algae and for rpoC1 of Chlamydomonas . While chlorophycean green algal cpDNAs were most affected by the abovementioned structural changes, their ulvophyte homologues were altered to an intermediate degree relative to the chlorophycean and Chlorella genomes [3, 4].
To gain insights into the evolutionary trends of the chloroplast genome in the Trebouxiophyceae and to elucidate the relationships among the various lineages of this class, we undertook the complete sequencing of cpDNA from phototrophic trebouxiophytes occupying lineages distinct from that represented by Chlorella and Helicosporidium (Chlorellales). We describe here the chloroplast genome sequence of Leptosira terrestris, a filamentous alga formally named Pleurastrum terrestre  and currently thought to belong to the Ctenocladales. Even though this genome resembles its Chlorella homologue in missing an IR, it is considerably shuffled in gene order and displays derived features that were previously observed in ulvophyte and/or chlorophycean green algal cpDNAs. Our results highlight the great plasticity of the chloroplast genome in the Trebouxiophyceae.
General features of Leptosira and other chlorophyte cpDNAs
Gene content and gene expansions
The chloroplast gene repertoire of Leptosira differs from that of Chlorella by the absence of three protein-coding genes (chlI, ccsA and ycf12) and three tRNA genes [trnL(gag), trnS(gga) and trnT(ggu)]. Although the latter three genes are missing, the set of 28 tRNA species encoded by Leptosira cpDNA is sufficient to read all codons present in this genome. The trnL(gag) and trnT(ggu) genes are also missing in the two ulvophyte and three chlorophycean green algal chloroplast genomes sequenced so far and chlI is absent from all three chlorophycean genomes: however, the ccsA and ycf12 genes have been retained in all these genomes. Aside from Leptosira, the trnS(gga) gene has been lost from all UTC algal cpDNAs previously investigated, except Chlorella and Stigeoclonium.
As in other UTC algal chloroplast genomes, a small fraction of the genes in Leptosira cpDNA have expanded coding regions relative to their Nephroselmis and streptophyte homologues. Nine genes in the Leptosira genome are more than 50% larger than their Mesostigma counterparts (cemA, ftsH, rpl19, rpoA, rpoB, rpoC1, rpoC2, ycf1 and ycf4) compared to only three in Chlorella cpDNA (cemA, ftsH and ycf1) . In addition, for the latter three expanded genes, we find a more important expansion factor in Leptosira than in Chlorella.
Like its chlorophycean green algal homologues, the chloroplast rpoB gene of Leptosira consists of two separate ORFs (rpoBa and rpoBb) that are not associated with sequences typical of group I or group II introns (Figure 1). As in Chlamydomonas and Scenedesmus cpDNAs, these ORFs are contiguous in Leptosira cpDNA and are separated by stop codons. Reverse transcriptase-PCR analysis failed to identify a genuine transcript encompassing both the Chlamydomonas rpoBa and rpoBb codingregions ; however, distinct transcripts were found to be specific to these ORFs . This result together with the observation that the Stigeoclonium rpoBa and rpoBb are encoded by different DNA strands and map to separate genomic loci  suggest that the two ORFs are transcribed independently. The Chlamydomonas rpoBa and rpoBb are considered to be functional genes, because no chloroplast-targeted RNA polymerase gene could be identified in the nuclear genome of this alga . The fragmentation of rpoB in Leptosira cpDNA and its chlorophycean homologues is reminiscent of the well-known case of rpoC gene fragmentation in cyanobacteria and plastids (rpoC1 and rpoC2) .
The 11 ORFs of more than 60 codons that we identified in intergenic regions of Leptosira cpDNA failed to display any similarity with known DNA sequences. All these ORFs differ from the conserved protein-coding genes at the levels of codon usage and their tendency to be richer in A+T.
Gene distribution between the two DNA strands
The propensity of adjacent genes to be located on the same strand is a property distinguishing all UTC algal cpDNAs, except the Chlorella genome, from Nephroselmis and streptophyte cpDNAs. The degree to which neighbouring genes are clustered on the same strand is reported in Figure 3 for various chlorophyte chloroplast genomes using the sidedness index (Cs) of Cui et al. . This index was calculated using the formula Cs = (n - n SB )/(n - 1), where n is the total number of genes in the genome and n SB is the number of sided blocks, i.e. the number of blocks containing adjacent genes on the same strand. When Cs reaches the maximum value of 1, all genes are located on one strand. In Figure 3, it can be seen that the sidedness index of Leptosira cpDNA (Cs = 0.88) is comparable to those of most other UTC algal cpDNAs. However, in contrast to Stigeoclonium cpDNA and Helicosporidium plastid DNA, analyses of cumulative GC and AT skews indicated that the coding strand bias in the Leptosira genome is not associated with a strand bias in base composition.
Only two derived gene clusters, trnV(uac)-trnL(caa) and rpl20-rps18, are shared specifically between Leptosira and Chlorella cpDNAs. The rpl20-rps18 gene pair is also conserved in the ulvophytes Oltmannsiellopsis and Pseudendoclonium.
Minimal numbers of inversions estimated in pairwise comparisons of gene order in chlorophyte cpDNAs
Number of inversionsa
Introns in Leptosira cpDNA and homologous introns at identical gene locations in other chlorophyte cpDNAs
Green algac/Intron numberd
Bryopsis plumosa (U)
Chlorella vulgaris (T)
Scenedesmus obliquus (C)
Chlorella vulgaris (T)
Chlamydomonas moewusii (C)
Scenedesmus obliquus (C)
Stigeoclonium helveticum i2 (C)
The most abundant repeated sequences in the Leptosira genome consist of dispersed repeats. Analysis of these repeats revealed two distinct groups of repeat units: repeat unit A with sequences of 25 bp (TTYAYCTGGGCAGGGAGATYYGRTC) and repeat unit B with sequences of 18 bp (CRGTWWATAAATCWWWGA). Each group of repeats features variants that differ slightly in primary sequence. Altogether, the 81 copies of repeat unit A and the 74 copies of repeat unit B represent 1.7% of the Leptosira genome sequence. In term of localization, a close relationship exists between repeat units A and B. Copies of repeat unit A are frequently found to be contiguous with the reverse complement of an almost identical sequence, creating imperfect palindromes. Copies of repeat unit B, in turn, are usually associated with such palindromes to generate larger palindromes of the type B-A-Arev-Brev, where rev stands for reverse complement. No repeats identical to the Leptosira repeat units A and B were detected in any other completely sequenced UTC algal cpDNA.
Multiple losses of the IR during the evolution of trebouxiophytes
As in Chlorella vulgaris cpDNA and Helicosporidium plastid DNA, we found that a rDNA-encoding IR is missing from the Leptosira chloroplast genome. Despite the absence of the IR in these three trebouxiophyte DNAs, there is little doubt that the chloroplast genome from the common ancestor of all trebouxiophytes featured a quadripartite structure very similar to that found in streptophytes and the prasinophyte Nephroselmis. This inference is supported by two separate observations. First, the partially sequenced chloroplast genome of Chlorella ellipsoidea, a representative of the trebouxiophyte order Prasiolales, displays a large IR, even though the latter region is atypical in containing a disrupted rDNA operon . Second, the IR-lacking Chlorella vulgaris cpDNA retains not only a remnant of an IR in the form of a pseudo rrs gene  but also the ancestral partitioning of genes displayed by prasinophyte cpDNA [3, 10].
Although the divergence order of the various monophyletic groups recognized in the Trebouxiophyceae remains ambiguous, the currently available phylogenetic data suggest that at least two distinct events of IR loss account for the disappearance of the IR in the three sequenced trebouxiophyte chloroplast genomes. The Trebouxiophyceae is a morphologically diverse assemblage that includes lichen phycobionts such as Trebouxia, free-living planktonic or terrestrial species, secondarily nonphotosynthetic coccoid algae and picoplanktonic coccoids [22, 23]. At least five distinct monophyletic lineages are recovered with 18S rDNA data [24–27], four of which correspond to the Trebouxiales, Microthamniales, Prasiolales and Chlorellales. Members of the Chlorellales, which include both Chlorella vulgaris and Helicosporidium, are consistently identified with high bootstrap support as the earliest-diverging branch of the Trebouxiophyceae, but the interrelationships among the remaining trebouxiophyte lineages remain ambiguous. This tree topology supports the view that the IR was lost independently in the Chlorellales and in the lineage leading to Leptosira. Obviously, for the Chlorellales, a single loss event is the most parsimonious explanation for the absence of the IR in Chlorella vulgaris cpDNA and Helicosporidium plastid DNA. To distinguish this scenario from the alternative hypothesis involving two independent losses, additional members of the Chlorellales will need to be surveyed for the presence/absence of this repeat.
In the light of previous reports indicating that loss of the chloroplast IR occurred relatively frequently during the evolution of the Viridiplantae, our inference that the IR was lost independently on at least two separate occasions in the Trebouxiophyceae does not imply that the quadripartite structure is less unstable in this algal group than in others. Aside from the Trebouxiophyceae, chloroplast genomes that experienced complete or almost-complete loss of the IR have been documented for the chlorophyte classes Ulvophyceae  and Chlorophyceae , for the charophycean lineage leading to the Zygnematales [29, 30] and for a number of land plants, including conifers and six tribes of legumes [31, 32]. Losses of the IR in conifers and legumes occurred independently and differed in the extent of the IR sequence lost, in the gene content of the IR prior to loss, and in the copy of the IR that was deleted . The site of deletion in pea cpDNA was found to exhibit duplicated gene fragments, but no simple mechanism involving recombination between these repeats could be postulated to account for the IR loss . In the present study, it was not possible to elaborate evolutionary models for the IR losses sustained by green algal cpDNAs, because the highly variable gene organization found in these genomes precluded inferences of gene order in ancestral IR-containing cpDNAs. Chloroplast genome sequences from more trebouxiophytes will thus be required to gain deeper insight into how the IR was deleted.
Similar evolutionary forces may have shaped the IR-lacking Leptosira and Stigeoclonium chloroplast genomes
We found that the Leptosira chloroplast genome differs considerably from its Chlorella vulgaris counterpart not only in gene order, but also in gene density, gene distribution between the two DNA strands and structure of some protein-coding genes. The important changes in gene order (Table 2) and in conservation level of ancestral gene clusters (Figure 4) observed for these trebouxiophyte cpDNAs are not surprising, given that IR loss is generally correlated with gene rearrangements [11, 12]. On the basis of this correlation, it has been hypothesized that IR loss enhances the frequency of intramolecular recombination between short dispersed repeats . In this context, it is worth mentioning that no short dispersed repeats = 30 bp with over 90% sequence identity are shared between the intergenic regions of Leptosira and Chlorella vulgaris cpDNAs, suggesting that these elements evolved independently in these two trebouxiophyte lineages. The fact that the Chlorella vulgaris genome displays a more ancestral gene order than its Leptosira homologue might be due to a more recent loss of the IR and/or a more recent proliferation of short repeats in the Chlorella vulgaris lineage.
Most intriguingly, the Leptosira chloroplast genome exhibits derived traits that are reminiscent of the evolutionary pattern observed for ulvophyte and/or chlorophycean cpDNAs. These derived traits were identified in the course of analyzing the following genomic features: (1) gene distribution over the two DNA strands, (2) gene density and (3) expansion and structure of protein-coding genes. The Leptosira chloroplast genes display a highly biased and asymmetrical distribution pattern over the two DNA strands, which most closely matches that observed for the chloroplast genome of the chlorophycean green alga Scenedesmus (Figure 3). The strong propensity of adjacent genes to be located on the same DNA strand in Leptosira cpDNA also mirrors the gene distribution patterns found in the chloroplast/plastid genomes of the two other chlorophyceans investigated (Stigeoclonium and Chlamydomonas), the ulvophytes Oltmannsiellopsis and Pseudendoclonium and the trebouxiophyte Helicosporidium. With regard to gene density, Leptosira cpDNA is currently known to be the most loosely packed chlorophyte genome (Table 1), followed by the Chlamydomonas and Stigeoclonium cpDNAs. The chloroplast genes exhibiting expanded coding regions relative to their Nephroselmis and streptophyte homologues are three-times more abundant in Leptosira than in Chlorella vulgaris, with the Leptosira set of nine expanded genes being more similar to those found in ulvophyte genomes with respect to coding content. In contrast to the conventional structure observed for rpoB in Chlorella vulgaris and ulvophytes, the Leptosira rpoB gene is fractured at the same site as that found for the fragmented genes of the three analyzed chlorophycean green algae (Figure 2). Therefore, two separate events of gene fragmentation, one occurring in the Leptosira lineage and the other before the emergence of the three chlorophycean groups examined so far, must be postulated to account for the distribution of the split rpoB structure among UTC algae.
From the similarities described above, it is tempting to propose that the same evolutionary forces shaped the IR-lacking chloroplast genomes in trebouxiophyte and chlorophycean lineages. However, considering the extraordinary fluidity of the chloroplast genome structure in the Chlorophyceae and the fact that no IR-containing chloroplast genomes from close relatives of Leptosira and Stigeoclonium have been investigated, it remains uncertain whether the common trends identified here are directly linked with the convergent events of IR loss that occurred in these chlorophyte lineages. For the Streptophyta, more specifically the zygnematalean lineages leading to Staurastrum and Zygnema, there exists convincing evidence that IR loss from the chloroplast genome was correlated with the expansion of intergenic regions and extensive gene rearrangements . Indeed, the low degree of compaction, the highly scrambled gene order and the numerous disrupted ancestral clusters observed in the Staurastrum and Zygnema genomes contrast sharply with the short intergenic spacers and with the extraordinary conservation of gene order and ancestral clusters exhibited by all their homologues in other streptophyte lineages.
The numerous derived features that we report here for the IR-lacking Leptosira chloroplast genome contrast sharply with the pronounced degree of ancestral features displayed by Chlorella vulgaris cpDNA, a trebouxiophyte genome also missing a rDNA-encoding IR. The close resemblance of the Leptosira genome with its ulvophyte and/or chlorophycean homologues with respect to the pattern of gene distribution, gene density and structure of protein-coding genes was also an unanticipated finding. On the basis of the current knowledge regarding the phylogeny of trebouxiophytes and the distribution of the presence/absence of the IR in the chloroplast genome, we conclude that the IR was lost independently in the Chorellales and the Leptosira lineage. The intriguing similarities between the derived features exhibited by the Leptosira chloroplast genome and those of its chlorophycean counterparts might suggest that the same evolutionary forces shaped the IR-lacking chloroplast genomes in the Leptosira and chlorophycean lineages. To test this hypothesis and better understand the dynamics of IR loss, IR-containing chloroplast genomes from close relatives of Leptosira and Stigeoclonium will need to be investigated.
Isolation and sequencing of Leptosira cpDNA
Leptosira terrestris (formally Pleurastrum terrestre Fritsch et John) was obtained from the University of Texas Algal Culture collection (UTEX 333) and grown in modified Volvox medium  under 12 h light-dark cycles. An A+T rich fraction containing cpDNA was isolated and sequenced as previously described . Sequences were assembled and edited with SEQUENCHER 4.2 (Gene Codes Corporation, Ann Harbor, MI). The fully annotated genome sequence has been deposited in [GenBank:EF506945].
Genes were identified by BLAST searches  against the nonredundant database of the National Center for Biotechnology Information server (NCBI) . Positions of ORFs and protein coding genes were determined using ORFFINDER at NCBI, programs of the GCG Wisconsin package (version 10.3) (Accelrys, San Diego, CA, USA) and applications from the EMBOSS version 2.9.0 package . Gene coding for tRNAs were localized with tRNAscan-SE 1.23 . The RpoB sequences were aligned using ClustalW 1.82 . Repeated sequences were identified with PipMaker  and REPuter 2.74 . Repeats were sorted with REPEATFINDER  and the retrieved classification was refined manually. Numbers of SDR units were determined with FINDPATTERNS of the GCG Wisconsin package version 10.3. The total length of genome sequences containing repeated elements was estimated with RepeatMasker  running under the WU-BLAST 2.0 search engine . Separate files containing the concatenated sequences of the intergenic regions of Leptosira and Chlorella cpDNAs were produced to search for the presence of shared repeated elements = 30 bp with up to 10% mismatches using the -d -p -l 30 -e 3 -seedlength 10 -q -v options of V match . The results of this analysis were visualized using GenAlyzer 0.81b .
Analyses of genome rearrangements
The GRIMM web server  was used to infer the minimal number of gene permutations by inversions in pairwise comparisons of chloroplast genomes. For these analyses, genes within one of the two copies of the IR were excluded from the data set and only the genes common to all compared genomes were analyzed. The data set used in the comparative analyses reported in Table 2 contained 86 genes; the three exons of the trans-spliced psaA and rbcL genes, the two exons of the trans-spliced psaC and petD genes, as well as the rpoBa and rpoBb genes, were coded as distinct fragments (for a total of 93 loci).
This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (to CL and MT).
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