ﬁve plastomes, respectively. Diﬀerences found comprise a relatively wide spectrum of changes, including additional or lacking predicted binding sites as well as single point mutations and spacing diﬀerences. Their functional relevance as that of transcriptional start sites remain to be veriﬁed. Regions coding for ribosomal binding sites were detected in 30 genes within the ﬁrst 23 bp upstream of the initial start codon by their match to the 30 end of the 16S rRNA. The sequences show no signiﬁcant diﬀerences among the plastomes. Evolutionary analysis, plastome pedigree Phylogenetic trees were generated by NJ, ML and MP using the 47 variable genes as well as 76 variable intergenic regions, and rooted by orthologous genes or sequence intervals, respectively, of the Lotus japonicus plastome sequence (19) as outgroup. All three methods grouped plastomes I, II and III in one clade with plastomes I and II being close relatives (bootstrap values 100% for NJ and ML) (Figure 4). ML supported a common ancestor for clade I–III and a clade consisting of IV and V (bootstrap 99%). MP resulted in one maximal parsimonious tree, placing plastome V in one clade with plastomes I–III, while plastome IV is located in a separate branch. This tree is supported both by NJ analysis of the concatenated alignments (bootstrap 96.7% for separation of plastomes V and IV) as well as by a species/consensus tree of the 47 individual NJ gene trees. In the consensus tree, only 28.9% result in a branching pattern identical to the ML topology, while 40% of all gene trees support a separate branch for plastome IV. Hence, we propose that the latter tree topology reﬂects the phylogenetic relationship between the ﬁve Oenothera plastomes (see ‘Discussion’ section). Average substitution rates of protein-coding genes between the ﬁve plastomes were investigated to derive an estimate of their divergence time. Average Ks values between the ﬁve plastomes varied over more than one order of magnitude, from 3 10 4 for plastomes I and II up to 3.6 10 3 for plastomes I/II and V. Applying a calibration derived for dicotyledoneous chloroplast genes (70), divergence for plastomes ranged generally between 416 000 (II and III) and 830 000 years. The most distant pair (I and V) has diverged approximately 1 million years ago, while divergence of plastomes I and II is estimated to be as recent as 83 000 years ago (Supplementary Table 6). All data obtained not shown in the manuscript are available upon request. DISCUSSION In this study we present the ﬁrst complete sequences of plastid chromosomes from closely related, morphologi- cally distinct and still interbreeding species, for which an organelle and nuclear genetics including cybrid technology is available. The populations of the North American subsection Oenothera are well characterized taxonomi- cally, geographically and ecologically for more than a Nucleic Acids Research, 2008, Vol. 36, No. 7 2373 Figure 4. Phylogenetic trees of the ﬁve Oenothera plastomes. Diﬀerent tree topologies appear depending on the method. NJ and MP place plastomes I, II, III and V in one clade and plastome IV in a separate branch (A), whereas ML puts I, II and III versus IV and V as two separate clades (B). century (5,21–24). Interspeciﬁc cybrids of the ﬁve geneti- cally distinguishable basic plastome types and the six basic genomes of that subsection can exist in all natural or artiﬁcial combinations (5,26 and Figure 1). The latter combinations are frequently developmentally impaired, revealing that co-evolution of nuclear and organellar genomes is an important element not only in speciation, but also in pre-speciation processes. In the clade diverging populations become apparent in changing chromosome formulas within species (22) and in intraspeciﬁc restriction fragment polymorphism of plastomes (29). The integrated compartmentalized genetic system of the plant cell, as of eukaryotic cells in general, evolves in its entirety generat- ing additional constraints to evolution not found in prokaryotes. A natural or an artiﬁcially introduced mutation in any one of the genetic compartments of the cell necessitates a compensatory event or has to fulﬁl the requirement that the entire system remains genetically balanced, fully functional and phylogenetically ﬁt (28). The coding potentials of the ﬁve plastomes, 113 unique genes, are nearly identical and comparable to those of plastid chromosomes of vascular plants in size, organiza- tion, gene clustering and conservation. They deviate from the ‘ancestral form’ of plastid chromosomes by a single kilobase-magnitude inversion in the large single-copy segment. Their sizes are moderately larger than those