The Leptosira chloroplast genome sequence assembles as a circular molecule of 195,081 bp encoding a total of 106 genes, not counting the intron ORF and the 11 free standing ORFs (Figure 1). All genes are present in single copy. Table 1 compares the general features of Leptosira cpDNA with those reported for the eight other chlorophyte cpDNAs completely sequenced to date. With an overall A+T content of 72.7%, the Leptosira genome ranks at the third position, after Helicosporidium plastid DNA and Scenedesmus cpDNA, with respect to the abundance of these bases. The 106 conserved genes, four introns and 11 free standing ORFs of more than 60 codons account for 48.3% of the total genome sequence of this trebouxiophyte, with the introns representing only 2.3% of the sequence. This is the lowest coding density among all examined chlorophyte cpDNAs. Intergenic regions have an average size of 981 bp, a value slightly higher than those observed for Stigeoclonium (950 bp) and Chlamydomonas (941 bp) cpDNAs. All four introns belong to the group I family.

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) 3. 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 9; however, distinct transcripts were found to be specific to these ORFs 9. This result together with the observation that the Stigeoclonium rpoBa and rpoBb are encoded by different DNA strands and map to separate genomic loci 7 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 15. 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) 16.

Figure 2 shows the results of our comparative sequence analysis of the translated rpoB sequences from Leptosira, the seven other photosynthetic chlorophytes whose chloroplast genome has been scrutinized, and two streptophytes. Regions displaying high sequence divergence and significant heterogeneity in size were identified at the two termini and at 11 internal sites. Some of the variable, internal regions represent insertions that are unique to one or more of the taxa examined. For example, the sequences mapping to three sites are present only in Leptosira, whereas that corresponding to a fourth site is specific to Stigeoclonium. Blast analyses conducted during the course of the present study revealed that the insertion sequence of 390 amino acids at the latter site displays features typical of inteins 17; this finding was made independently by another laboratory and documented in InBase 1718 under the accession name She_RPB2. The rpoB genes of Leptosira and of the three chlorophycean green algae are fragmented at the same site, near the junction of a conserved segment of 80 codons and a highly variable region (ranging from 7 to 803 codons in size) found in all taxa.

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.

With 78 genes occupying one strand and 28 genes the other strand, the gene distribution over the two DNA strands of the Leptosira genome is highly biased (Figure 1). While a similar bias has been observed for Scenedesmus cpDNA (77 genes on one strand and 26 genes one the other) 8, other completely sequenced chlorophyte cpDNAs display about the same number of genes on the two strands (Figure 3). All 78 Leptosira genes found on the same cpDNA strand, except ycf62 and cemA, are grouped together, forming four separate stretches containing 9 to 24 genes.

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 (C_s) of Cui et al. 13. This index was calculated using the formula C_s = (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 C_s 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 (C_s = 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.

Unlike its Chlorella homologue, the Leptosira chloroplast genome does not reveal any obvious remnant of the ancestral gene partitioning pattern displayed by Nephroselmis and most streptophyte cpDNAs (Figure 1). However, the two trebouxiophyte genomes resemble one another with respect to the conservation of ancestral gene clusters (Figure 4). Leptosira cpDNA exhibits nine of the 24 ancestral clusters conserved between Mesostigma and Nephroselmis cpDNAs as well as the remains of five other clusters; altogether, these conserved clusters encode 53 genes. In Chlorella, 62 genes are found to be part of 11 intact and four partially conserved, ancestral clusters. Breakage of three of the intact clusters present in Chlorella cpDNA, further fragmentation of the four partially conserved clusters in this alga and specific retention of an ancestral cluster [rpl20-trnD(guc] absent from the Chlorella and other UTC cpDNAs explain the main differences observed in the Leptosira genome. Note that these rearrangement events involved the disruption of the highly conserved rRNA operon [rrs-trnI(gau)-trnA(ugc)-rrl-rrf] downstream of rrs and the relocalization of this gene 62 kb away from the remaining portion of the operon.

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.

To compare the degrees of similarity in gene order between the Leptosira and other green algal cpDNAs, we also estimated the minimal number of gene permutations that would be required to convert the gene order of a given genome to that of another genome. More specifically, we examined the orders of the 86 genes common to the cpDNAs of Leptosira, Chlorella, Nephroselmis, Oltmannsiellopsis, Pseudendoclonium, Stigeoclonium, Scenedesmus and Chlamydomonas using GRIMM 19. We found that the Leptosira and Chlorella genomes differ by as many as 53 inversions. As shown in Table 2, this level of gene rearrangements is identical to that found for the cpDNAs of the ulvophytes Oltmannsiellopsis and Pseudendoclonium but lower than those obtained for the chlorophycean algae Stigeoclonium, Scenedesmus and Chlamydomonas (59–83 inversions). Furthermore, our results showed that the Nephroselmis, ulvophyte and chlorophycean genomes, except Stigeoclonium cpDNA, display more similarity in gene order with the Chlorella genome than with Leptosira cpDNA.

The four group I introns in Leptosira cpDNA interrupt chlL, psaB, psbC and trnL(uaa). They fall within two different subgroups (IA1 and IC3), with the IA1 subgroup including the three introns found in protein-coding genes. All four introns, except that present in psbC, are positionally and structurally homologous to previously reported introns in chlorophyte cpDNAs (Table 3). In this context, it is worth noting that Leptosira and Chlorella cpDNAs share homologous introns in trnL(uaa) and chlL. The psbC intron is unique to Leptosira and displays an ORF. The predicted protein of 415 amino acids specified by this ORF is related to the LAGLIDADG endonucleases and contains two copies of this motif.

Comparison of the Leptosira cpDNA sequence against itself reveals that short repeats are present in many intergenic regions as well as in some introns and genes (predominantly expanded genes) (Figure 5). Repeats represent 5.1% of the total genome sequence and 8.5% of the total size of the intergenic regions. The relative abundance of these elements is therefore lower than in Chlorella cpDNA (7.8% of total genome and 14.9% of intergenic regions) but is comparable to those observed in Scenedesmus and Pseudendoclonium cpDNAs (see Table 2 in 20). In all other completely sequenced cpDNAs of photosynthetic UTC algae, short repeats account for more than 10% of the genome. The longest repeated sequence in Leptosira cpDNA is 194 bp in size, whereas the maximal size of the Chlorella repeats is two-fold smaller (84 bp).

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-A_rev-B_rev, 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.

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 21. Second, the IR-lacking Chlorella vulgaris cpDNA retains not only a remnant of an IR in the form of a pseudo rrs gene 5 but also the ancestral partitioning of genes displayed by prasinophyte cpDNA 310.

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 2223. At least five distinct monophyletic lineages are recovered with 18S rDNA data 24252627, 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 28 and Chlorophyceae 7, for the charophycean lineage leading to the Zygnematales 2930 and for a number of land plants, including conifers and six tribes of legumes 3132. 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 32. 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 33. 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.

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 1112. On the basis of this correlation, it has been hypothesized that IR loss enhances the frequency of intramolecular recombination between short dispersed repeats 31. 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 30. 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.

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 34 under 12 h light-dark cycles. An A+T rich fraction containing cpDNA was isolated and sequenced as previously described 35. 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 36 against the nonredundant database of the National Center for Biotechnology Information server (NCBI) 37. 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 38. Gene coding for tRNAs were localized with tRNAscan-SE 1.23 39. The RpoB sequences were aligned using ClustalW 1.82 40. Repeated sequences were identified with PipMaker 41 and REPuter 2.74 42. Repeats were sorted with REPEATFINDER 43 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 44 running under the WU-BLAST 2.0 search engine 45. 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 46. The results of this analysis were visualized using GenAlyzer 0.81b 47.

The GRIMM web server 19 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).