repetitive regions and are highly variable between all species. RESULTS The plastid chromosomes of the ﬁve Oenothera species that represent the genetically distinguishable basic plas- tomes of subsection Oenothera were completely sequenced on both strands using a primer-based strategy. The sequences are deposited in GeneBank under the accession nos. AJ271079.3, EU262887, EU262889, EU26890 and EU262891. They include a revised version of plastome I of Oe. elata subsp. hookeri strain johansen (see ‘Materials and Methods’ section) and conﬁrm the corrected ycf2 sequence reported recently (41). A corrigendum of the previous plastome I data will appear (30). Size, gene content and design of the Oenothera plastid chromosomes The individual plastome sequences assemble as circular structures of 165 728 bp (plastome I), 164 807 bp (plastome II), 165 225 bp (plastome III), 163 365 bp (plastome IV) and 165 055 bp (plastome V) in size including 56.6% coding regions and 43.4% non-coding regions (spacers and introns). The overall G+C content is 39.1%, about 41.7% in coding and 36.5% in non-coding sequence intervals (Supplementary Table 1), and is thus somewhat higher than found usually (36.8–39.0%) (http:// www.ncbi.nlm.nih.gov/genomes/ORGANELLES/plastids _tax.html). As expected, the chromosomes display the typical quadripartite anatomy (40,58) characteristic for many plastid chromosomes, i.e. a pair of large inverted repeats that separate two single copy regions, the large single copy (LSC) region and the small single copy (SSC) region. The overall divergence of the chromosomes is expectedly low, between 96.3% and 98.6% similarity and 96.1% to 98.5% identity (Supplementary Table 2), and comparable to that found among various Nicotiana species and Atropa (96.0–98.5 identity; 10,11). The ﬁve Onagracean chromosomes encode an identical set of 113 unique genes with identical gene order and gene clusters, 17 of these are duplicated in the IR (Figure 2). Genes, gene order and gene clusters are essentially colinear with those in the chromosomes from Nicotiana (10), Lotus (19), Atropa (11), spinach (59), Arabidopsis (60) and Eucalyptus (61) except for a large inversion of 56 kb in the LSC region (30,40,62,63) that occurred in the intergenic regions between the accD/rbcL and rps16/ trnQUUG and reverses the order of genes between rbcL and trnQUUG (Figure 3). Speciﬁc also for the Oenothera plastid chromosomes are two copies of the initiator tRNA trnfMCAU which diﬀer by a single nucleotide polymorphism in plastomes I, II, III and IV and are part of a tandem repeat structure (see later). The gene complement of the Oenothera plastid chromo- somes is comprised of 4 rRNA genes (16S, 23S, 5S, 4.5S), a total of 31 distinct tRNA genes (the rrn genes and 7 trn genes are duplicated in the IR) and 78 protein-coding loci including ycf1, ycf2, ycf3 and ycf4 (Supplementary Table 3). Sixteen genes contain a single intron, one gene Nucleic Acids Research, 2008, Vol. 36, No. 7 2369 (ycf3) contains two; as in other species, rps12 mRNA is generated by transsplicing. All ﬁve species share the same set of introns, one class I intron (trnLUUA) and 17 of class II. Compared to tobacco, clpP lacks both introns, even though the surrounding coding sequences are conserved. In both cases, the deleted sequences coincide precisely with the established intron borders so that the mechanism of intron loss may have involved a processed RNA intermediate. As in various other plastid chromo- somes, two pairs of genes overlap, atpB-atpE (4 bp) and psbC-psbD (52 bp); matK is located within the intron of trnKUUU. The 31 tRNA species (including both copies of trnfMCAU) represent 20 amino acid species and are suﬃcient to satisfy all the requirements for protein synthesis in the organelle. All protein-coding genes use the standard plastid/bacterial code with a predicted methionine/ATG start codon, except of ndhD which starts with an ACG [that is edited as has been shown for plastid chromosome I and IV (64)] and cemA which has been tentatively annotated with an ATA start codon (Supplementary Figure 1). The most common stop codon is TAA (51.3%) as in other plastid chromosomes (65). Seventy-three of the 78 protein-coding genes are present in most angiosperm plastomes. Five, accD, rpl22, rpl23, ycf1 and ycf2, are not generally found, but appear to be functional in Oenothera. Ycf1 and ycf2 with at least 2360 and 2313 codons in length, respectively, are well conserved within all studied dicotyledoneous plants; the former is entirely located within SSC. InfA, present in various species, appears to be a pseudogene in all plastomes, as in tobacco, Arabidopsis and Eucalyptus. SprA, reported from various dicots, e.g. Solanaceae, Arabidopsis or spinach (11,66), is missing. The IRA/LSC, LSC/IRB and IRB/SSC junctions are identical in all ﬁve plastomes; the SSC/IRA junction is identical in plastomes I, II and III, but diﬀers by 2 additional bp in plastomes IV and V. The region downstream of ycf1 until ndhF0 is highly polymorphic among all ﬁve plastomes. NdhF and rps19 at the border of IRB generate truncated versions at the SSC/IRA and IRA/LSC junctions, respectively. NdhF0 lacks the 50 end, rps190 the 30 end. It is not known whether the truncated versions are functional. Another region with locally conserved homology, noted pre- viously in plastome I and often designated as ycf15, is present in all plastomes. Since it has premature in-frame stop codons generated by frequent insertions of variable sizes and no function or corresponding protein is known (59) we excluded it from the list of genes (Supplementary Table 3). Sequence divergence and sequence repetition in Oenothera plastomes The ﬁve plastomes are perfectly syntenic. This allowed a comparative analysis of indels and sequence repetition, which are usually diﬃcult to analyze between distant species. Insertions, deletions and repetitions are relatively frequent. Relative to plastome IV, an additional 1456 nt insertions and 3819 nt deletions are found in plastome I. The corresponding nt numbers for the plastomes II, III and V are 1156/2598, 1701/3561, 864/2557, respectively.