2374 Nucleic Acids Research, 2008, Vol. 36, No. 7 found generally for higher plants and belong to the largest plastid chromosomes known from vascular plants that usually range between 130 and 160 kb (reviewed in 71). Genes are well-deﬁnable, except of the N-terminus of cemA (ycf10) encoding an inner envelope polypeptide involved in CO2 uptake, even after comparison with cemA loci from 50 reference species in which N-termini are not consistent (Supplementary Figure 1). Judged from PCR and Western analysis, the locus does not appear to be a pseudogene; also no evidence was found for a nuclear copy of this gene. This point remains to be settled in general. Despite their gross conservation and close relationship, Oenothera plastid genomes are remarkably diverged, compared, for instance, to those of the Solanacean genera Atropa belladonna and tobacco (11). All four kinds of mutations that have been reported from plastid chromosomes, point mutations including changes in restriction sites, repetitions, insertions/deletions and inver- sions occurred. The former are scattered all over the circular chromosome, but found with diﬀerent frequency in the individual sequence classes (Figure 3). Somewhat less mutations are expectedly found in the IR, probably due to copy-correction of the repeated segments (e.g. 72). Insertions/deletions and repetitions in direct or inverse orientation and restricted to repeat sizes of 16/30 bp or larger with >90% sequence identity are relatively frequent (30,73). Repetitive and divergent regions are correlated suggesting a role of repeats in generating divergent regions, e.g. by illegitime recombination. Slipped mispair- ing may cause or contribute to changes in a number of instances as well (73–75). Coding sequences are generally well-conserved among the plastomes. Eight genes, almost exclusively non-photosynthetic, diﬀer by indels, nearly a dozen by repeats including plastome-speciﬁc changes. Although non-protein and non-RNA coding regions, such as intergenic sequences and introns (together amounting nearly 45% of the chromosomes) evolve faster than genes, they still are quite well-conserved commensurate with the close relationship of plastomes and indicative that functional elements within these sequence intervals are conserved and compactly bundled. The junctions of the IR regions that often cause size diﬀerences between sperma- tophyte plastid chromosomes (e.g. 76) are almost invariant. Inversions of diﬀerent sizes in plastid chromosomes have been reported from various taxa including Bryophyta, gymnosperms, grasses, Fabaceae, Ranunculaceae, Caryo- phyllaceae, Geraniaceae, Asteraceae, Campanulaceae and Onagraceae (http://www.ncbi.nlm.nih.gov/genomes/ ORGANELLES/plastids_tax.html). Three points of gen- eral interest are noteworthy. (i) Features that have been proposed to mediate inversions in plastid chromosomes by homologous or illegitimate, inter- or intramolecular recombination include the presence of dispersed repeated sequences and/or tRNA genes which frequently lie at or adjacent to endpoints (18,77–80). However, none of the elements discussed appears to extend to all evolutionary rearrangements and no satisfactory general model or explanation which would account for their involvement is available. In Oenothera, only one of the junctions lies adjacent to a tRNA gene. The exact breakpoints delimiting the inversion between trnQ and rps16 on the one end and accD and rbcL on the other (63) cannot be assigned by sequence comparison with the non-rearranged berteriana plastome, because of the complex pattern of repetitive sequences, of a lack of readily discernible vestiges of sequences involved bordering inversions, and because causes and mechanisms of the structural alteration that might reveal footprints of the event are not really under- stood. Single copies of sequences that are repetitive, scrambled and arranged in a relatively complex way in Oenothera are detectable in the Munzia plastome. Whether the structural change is in any way directly related to these elements remains to be shown. (ii) Mapping of polymorphic restriction fragments in Oenothera plastomes has provided evidence of ﬁve preferred sites of mutation (40) which coincide well with sequence data and of which two may be recombinogeni- cally relevant, since they reside at or near the inversion points (Figure 3). An intriguing number of inversions in diﬀerent materials occurred at nearly identical positions, including these sites e.g. (15,18–20,78,79,81–83). For instance, a 50 kb inversion reported from legumes (19,20,83) resembles, but is not identical, to that found in Oenothera since it includes rps16 besides trnQ at one of the junctions. Two of the three inversions in Adonis and polymorphism in some grasses include the same region (15,79). These mutational ‘hot spots’ may be shared with the non-rearranged plastid chromosomes of tobacco species (84) and restriction analysis indicates that at least some of them contribute to intraplastome variation in Oenothera, which has been noted from all ﬁve plastomes (29 and unpublished data). Intraplastome variation is not rare (80,85), genetically virtually neutral, but the relevance of individual events in speciation, remains to be veriﬁed. In Oenothera, it would be particularly interesting to see whether the patterns reﬂect solely mechanistic imprecision with no phylogenetic relevance or correlate in part with geographic trends and genetic drifts of subpopulations in species diﬀering in their chromosome formulas (22,24). Whatever causes and relation to recombinatoric events, it is unlikely that the shared sites reﬂect random eﬀects. The underlying processes are probably comparable and of general nature, and, at least in some instances, may involve further features, such as transcription, some sequence speciﬁcity, elements of secondary structure or sites resembling att-lambda (86,87). Obviously, structural changes in plastid chromosomes are more complex than presently anticipated. An understanding of the require- ments and modes of recombination could presumably contribute to clarify functional and evolutionary processes of plastid chromosomes, possibly also some way of transposition of sequence intervals to other organelles, but this can probably not be achieved by mere sequence study and will require more penetrating approaches, such as transformation strategies to determine recombinogenic activity of distinct constructs in vivo before general principles become apparent. (iii) The Oenothera inversion disrupts known transcrip- tional linkage. Disruption of operons in plastid DNA of vascular plants by structural rearrangements is considered