to be rare; only a few cases have been postulated from legumes and Campanulaceae (79,88–90). However, at present this inference is not fully conclusive due to the recent demonstration of a second plastid located RNA polymerase of nuclear origin (NEP, 67) which has not yet been considered in rearrangement analysis. As the ancestral eubacterial RNA polymerase type PEP, the phage-type NEP polymerase reads the entire plastid chromosome (47) though from diﬀerent, often multiple and even operon-internal promoters (91). The Oenothera inversion separates accD from rbcL which in non- rearranged plastid chromosomes are members of a larger gene cluster, including rbcL-accD-psaI-ycf4-ycf10-petA. In tobacco, spinach, Arabidopsis and maize, this cluster is co-transcribed from a PEP promoter upstream of rbcL into a giant RNA species of 9 kb that is subsequently processed, often rapidly (Figure 5; 92). Removal of rbcL from the operon either required the replacement of the rbcL promoter by that of trnQ, use of the operon-internal NEP promoter in front of accD (93), acquisition of a new PEP promoter 50 to accD (or trnQ) or use of the NEP promoter upstream of rpoB (c.f. Figure 2). The latter alternative is not likely, because of the general weakness of that promoter. On the other hand, the translocated rbcL promoter may serve the rbcL-rps16-trnKUUU-matK- (psbA-trnHGUG) gene cluster, since an RNA species of corresponding size can be detected in Oenothera (Figure 5). Obviously, the promoter patterns of both regions need to be functionally deﬁned to settle the transcriptional consequences of the inversion. At the generic level the large inversion shared among all Oenothera plastomes provides a robust phylogenetic marker. The almost identical endpoints and its absence in the closely related South American subsection Munzia, in subsection Raimannia (62) or in the sister group Epilobium (94) suggest that this inversion is not caused by one of the proposed rare parallel inversions (79–81), but has arisen monophyletically within the Oenothera clade and late in the history of the Onagracean complex. It predates the divergence of the subsection, occurred either in the basic plastome IV or in the common ancestor of the subsection, and hence marks a recent split in the history of the genus. Even if the absolute calibration with less than 1 million years based on the molecular clock may require correction, the more than 10-fold diﬀerence of divergence times of the ﬁve plastomes will remain (Supplementary Table 6). The plastome cladograms determined separately from protein-coding and intergenic regions are nearly congruent and conﬁrm the pedigree deduced from formal genetic approaches on the basis of both compartmental compatibility relations to genomes and diﬀerences in multiplication rates of plastids which are independent traits (5,73,95). The genetic determinants for the latter feature reside predominantly in the plastome (96). The pedigree is also consistent with changes in the thylakoid proteome mentioned earlier, notably the psbB variance of plastomes IV and V and that of atpA of plastome III (29 and Supplementary Figure 4), with a 2 bp IR extension shared by plastomes IV and V as well as with various other characters. Nucleic Acids Research, 2008, Vol. 36, No. 7 2375 Figure 5. Northern analysis of rbcL operons from rearranged Oe. elata subsp. hookeri (A) and non-rearranged spinach (B) plastid chromosomes. Sequences of entire plastid chromosomes have become a powerful research tool, not only to probe into chloroplast biogenesis, function and engineering, but also into eukaryotic genome evolution. The genus Oenothera provides unique opportunities to develop phylogenetic analyses based on mere sequence data into a ‘functional molecular phylogeny’ by applying other kinds of approaches, such as formal genetics combined with those of molecular and cell biology or physiology, to study diversiﬁcation of populations and speciation pro- cesses in wider perspective. The availability of complete Oenothera plastome sequences should facilitate access to unveil molecular mechanisms that contribute to plant genome evolution, speciﬁcally to compartmental co-evolution, leading to divergence of populations or new species. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS This work was supported by Grants from the Deutsche Forschungsgemeinschaft (SFB-TR1) to R.G.H. and J.M., and by the Hanns-Seidel-Stiftung (Bundesministerium fu¨ r Bildung und Forschung) to S.G. We highly appreciate the participation of Rainer Maier who passed away in the early phase of the project. We thank Elli Gerick and Ingrid Duschanek for their excellent technical assistance, Peter Poltnigg and Elena Funk for help in sequence evaluation and Holger Gru¨ ne for the spinach Northern blot. The CemA antiserum was kindly provided by Ju¨ rgen Soll. Funding to pay the Open Access publication charges for this article was provided by the Deutsche Forschungsgemeinschaft (SFB-TR1). Conﬂict of interest statement. None declared. REFERENCES 1. Herrmann,R.G. (1997) Eukaryotism, towards a new interpretation. In Schenk,H.E.A., Herrmann,R.G., Jeon,K.W., Mu¨ ller,N.E. and