2368 Nucleic Acids Research, 2008, Vol. 36, No. 7 BLAST algorithm (44,45) provided by the National Centre for Biotechnology Information (Bethesda, MD, USA). Assembly and evaluation of sequences were performed with the SeqMan 6.1 program (DNASTAR Inc., Madison, WI, USA) using plastome I (accession no. AJ271079) as template for the other plastomes. All plastome sequences were aligned using the MegAlin 6.1 program (DNASTAR Inc., Madison, WI, USA) and the program BioEdit 5.0.9 (North Carolina State University) as alignment editor (46). Gene annotation of plastomes II to V was guided by the annotated plastome I (30). Verification of inversion breakpoints Total DNA of Oe. villaricae strain berteriana Schwemmle [syn: berteriana Erlangen (47)] was isolated as described earlier. The primer pair rbcLfor (50-TGTGGCATATGCC TGCTCTG-30) and psaI_IVP11rev (50-GGAGAAATCC ATTCTTGTCGTC-30) deduced from a highly conserved region in the Oenothera plastomes was used to sequence the interval equivalent of the inversion breakpoint between rbcL and accD in the berteriana plastome. PCR was performed with BIO-X-ACTTM Long DNA Polymerase (Bioline GmbH, Luckenwalde, Germany) according to the standard protocol and the product was sequenced. The same strategy was used with the conserved primer pair rps16_IIP3for (50-GAACAGAAGAAAGGG TGTCGAG-30) and trnQ_IVP37for (50-CACTGGAATT GACGAATAACC-30) for the corresponding region of the inversion between rps16 and trnQ. Northern analyses Repeat analysis Northern analysis was performed as described (48). Two diﬀerent types of repeats, palindromes and direct tandems, were analyzed in this study applying the programs palindrome and etandem of the EMBOSS suite (49). For both repeat types, the minimal cut-oﬀ identity between two copies was set to 90%. In case of multiple copies for one tandem, each copy was required to have at least one other member matching this constraint. The minimal and maximal copy size investigated were 16 to 100 bp for palindromic and 10 to 100 bp for tandem repeats, respectively. Gap size between palindromes was restricted to a maximal length of 3 kb. Overlapping repeats with sequence similarity were grouped into one repeat motif. For palindromes, both the direct and inverted part of two repeats had to overlap. For each repeat motif, the longest element is provided as its representative. Inversion breakpoints were separately analyzed in the ﬁve plastomes of the subsection Oenothera and berteriana Schwemmle. Computational prediction of sigma-factor and RNA polymerase binding sites Multiple alignments of intergenic regions delimited either by the 50 neighboring gene or a maximum size of 600 bp were searched for polymerase binding sites. Due to over- lapping binding speciﬁcity of diﬀerent bacterial polymer- ase-like (PEP) s-factors in plastids (50), no attempt was made to diﬀerentiate between individual factors. The consensus sequence TYRMNN(N)16–20 WANNWT was selected as search pattern which covers a wide range of experimentally reported sites. The regular expression found was similar to but less speciﬁc than the consensus suggested by Homann and Link (51) and Kanamaru and Tanaka (52). Binding sites of the phage-type polymerase (NEP promoters) were deﬁned as matches to the consen- sus ATA0–1N0–1GAA(N)15–23YRT (53,54) representing NEP type Ib promoters. The other two NEP promoter types have been excluded from the analysis as the consen- sus of NEP type Ia promoters—YRTa—is too low for computational predictions and NEP type II promoters are known from just a single case (50). Candidate binding sites were positioned within the multiple alignments and edited by manual supervision to correct for misaligned regions, e.g. due to small repeats. Prediction of Shine–Dalgarno sequences Sequences 50 bp upstream of the start codon were searched for candidate Shine–Dalgarno regions using the program free2bind (55) and the 30 16S RNA sequence of Oenothera. A minimum free energy of 4.4 kcal and a maximum distance to the start codon of 23 bp was required for the reported matches. Evolutionary analyses Phylogenetic trees were generated from concatenated multiple codon-based alignments of the 47 genes variable in the ﬁve Oenothera plastomes. The dataset comprises 44 472 aligned characters present in six species including corresponding sequences of the Lotus japonicus plastome (19) as outgroup for tree rooting. Neighbor-Joining (NJ), Maximum-Likelihood (ML) and Maximum Parsimony (MP) from the PHYLIP package (56) were applied to infer trees. Bootstrap analysis for NJ and ML was performed with 1000 random samples each. In addition, gene-speciﬁc phylogenetic trees for all variable genes were determined by NJ and ML. A species tree was then built from individual gene trees using the consense program of PHYLIP. Trees for non-coding sequences were derived from 76 intergenic regions which showed nucleotide substitutions between the ﬁve Oenothera plastomes. Synonymous and non-synonymous substitution rates were estimated applying the yn00 programme of the PAML package (57). F3x4 were selected as substitution matrix and Ka and Ks were determined by the Nei–Gojobori method as implemented in yn00. Rates for protein-coding genes variable among at least two of the ﬁve Oenothera plastomes were estimated from pairwise codon-based alignments. For ﬁve diﬀerent plastomes, there are 10 pairwise combinations for each gene, resulting in a total of 780 rates for all and 470 rates for variable genes. Note that the computation of o: = Ka/Ks is not always applicable (e.g. for Ks = 0). Therefore, o: could be determined for only 215 pairwise combinations. To compare average Ka and Ks rates between species, a concatenated alignment of individual protein-coding regions were analyzed. Ycf1, ycf 2 and accD were excluded from the analysis as they contain large portions of