2372 Nucleic Acids Research, 2008, Vol. 36, No. 7 two tandem repeats in plastome IV and none in plastome V. Three palindromic repeats were detected at the rps16/ rbcL junction of plastomes I and II. In the trnQUUG/accD spacer region between two and no palindrome(s) were identiﬁed. Of particular note are the elements of clustered palindromes split among both spacer regions which were detected in all ﬁve plastomes. Their number varies enormously, between 1 (plastome V) and 17 (plastome III). Also, the same sequence motifs can be present in tandem and palindrome arrangement. The repetition patterns preclude an accurate demarcation of the insertion breakpoints and also aﬀect the highly variable N-terminal region of AccD (Supplementary Figure 1 and see later). In an attempt to better understand the inversion breakpoints and underlying processes, the corresponding regions in Oe. villaricae strain berteriana Schwemmle, a member of the closely related sister subsection Munzia, were sequenced (rps16/trnQUUG, accession no. EU255777 and rbcL/accD, accession no. EU255778). Subsection Munzia lacks this inversion (62). The berteriana regions corresponding to the Oenothera breakpoints between rps16/trnQUUG and rbcL/accD do not display pronounced divergence. The spacer region between rps16 and trnQ in that plastid chromosome lacks tandem and palindrome repeats. Nevertheless, the entire region is conserved and present in two parts in all ﬁve Oenothera plastomes, separated by an interspersed Oenothera-speciﬁc sequence interval. The berteriana region between rbcL and accD, in turn, lacks palindromes, but contains two tandem repeats. Approximately 1.5 kb of the berteriana rbcL/accD spacer are unique to the berteriana plastome. Conversely, trnQ/ accD spacer sequences between 1.5 and 2.5 kb depending on the Oenothera plastome have no equivalent in the berteriana plastome. The same holds true for the Oenothera rps16/rbcL spacer, in which the number of unique nucleotides diﬀers between approximately 50 and 500. The repeat structure in the rps16/rbcL spacer of the Oenothera plastomes appears to be linked to the inversed arrangement of rbcL and the result of duplication and relocation during the inversion process, as the berteriana equivalent is missing. As mentioned, palindrome repeat copies split among both spacers are not rare in the Oenothera plastomes, but no such cases were detected in berteriana with the selected threshold. Differences in coding regions among Oenothera plastomes I–V Three distinct sequence classes, protein-coding loci, tRNA and rRNA genes, have been evaluated for sequence conservation and changes between the ﬁve plastomes. Among the 78 protein-coding genes identiﬁed (including ycf1 to ycf4), 31 are identical in their nucleotide sequence, while 47 are variable between at least one plastome pair (Supplementary Tables 4 and 5). All carry substitutions of individual nucleotides and eight additionally indels. Sixteen genes, namely atpE, cemA, ndhI, ndhJ, petA, petB, psaB, psaC, psbC, rpl16, rpl33, rpoC1, rps4, rps11, rps14 and rps19 contain only synonymous substitutions which do not change the coding context. Only non- synonymous substitutions causing amino acid changes were detected in 11 genes: ndhB, ndhC, ndhH, psbB, rpl32, rpoA, rpoB, rpoC2, rps2, rps8 and rps15. Rpl22 contains synonymous substitutions and a single bp insertion/ deletion, ndhF synonymous substitutions and a multiple base pair indel, rps18 multiple indels and a non- synonymous substitution. Both types of substitutions are found in ndhD, in plastome I together with a single base pair insertion, and a multiple base pair insertion in plastome V. Both, synonymous and non-synonymous substitutions are present in the remaining 16 genes (accD, atpA, atpB, atpF, ccsA, clpP, matK, ndhA, ndhE, petD, psaA, psbA, rps3, ycf1, ycf3 and ycf4). Five of them, clpP, accD, ndhD, ycf1 and ycf2, diﬀer by multiple indels among plastomes. Of the 31 tRNA genes, 30 are identical and 1, trnfMCAUII, is variable in 1 nt. The mutation is not part of the anticodon, should have no or only a negligible eﬀect on the folding of the tRNA, and hence not inﬂuence function, last not least since a second, not mutated copy, trnfMCAUI, is present in the Oenothera plastomes. The four rrn genes are identical in all ﬁve Oenothera plastomes. Protein-coding genes with length polymorphisms can be operationally grouped into three categories, those with reading frame shifts (ndhD, rpl22 and rps18), those without reading frame shifts (ycf1, ycf2, accD, clpP and ndhF) and a third class, including atpA and psbB, which are known to diﬀer in electrophoretic mobility of their products in plastome III and plastomes IV and V, respectively, independent of the genotype with which the plastid types are associated with (29). As most of the diﬀerences described reside in polypeptide regions known to be highly variable in plastid chromosomes in general, often at the very C-terminus of a polypeptide, it is unlikely that they are of functional and/or evolutionary relevance. Therefore, details of the changes in these 10 loci including veriﬁcation by SNP, CAPS, Western (cemA) analysis and comparison with corresponding loci of usually 4–8 reference species are presented in Supplementary Figures 1–4 and Supplementary Table 5. Differences in intergenic regions among plastomes I–V Intergenic sequence intervals, which house various func- tional elements, such as promoters, terminators, proces- sing signals or ribosome binding sites, are less conserved among plastomes (76 variable, 37 identical). On average, for each of the 113 intergenic regions, 24.1 indels (2743 for all regions) and 5.4 substitutions (616) have been detected. This contrasts observations made for coding regions in which only few indels have been found. Approximately 600 bp upstream of each gene were searched for potential promoters of the bacterial type ( 10 and 35 boxes) and phage-type polymerases. Various sites previously reported from other species (51,53,67–69) were found in the Oenothera plastomes. Seventy-ﬁve genes displayed a putative sigma-factor binding site and 69 genes contained type Ib-like NEP promoters. In total, for 88 genes at least one polymerase binding site could be deduced and in 56 genes promoter predictions were found for both types. For predicted PEP and NEP sites, 39 and 27 promoters had at least one diﬀerence in the binding site between one of the