in the development of the resulting hybrids or cybrids (5,6). Compartmental co-evolution generates Dobzhansky– Muller incompatibilities responsible for reproduction barriers and consequently represents an important, often neglected, factor in speciation processes (7,8). Combined with cybrid technology and transplastomic approaches, sequence data of entire plastid chromosomes from closely related species could provide access to molecular mechanisms underlying plant genome evolution (6,9). However, such data are still scarce. Some informa- tion is available from the Solanacean family (10–13), from a few related cereal species, notably maize, rice, wheat, barley and sugar cane (14–18), and from a few legumes (19,20). These data have led to reﬁned taxon topologies in some cases, and a recent study of the interspeciﬁc plastome–genome cybrids between tobacco and Atropa has allowed identiﬁcation of a ﬁrst molecular mechanism that causes plastome–genome incompatibility and could contribute to speciation processes (6,9). Ultimately, such approaches are meaningful only in a context in which the relationships of the plant groups being considered are well understood and other strategies, notably formal nuclear and organelle genetics, can be incorporated to overcome limitations of traditional phylogenetic analyses. The genus Oenothera is uniquely suited for studying diversiﬁcation of populations and speciation processes (5,21–25), because it possesses a unique combination of genetic features, and a comprehensive taxonomy, cytoge- netics, ecology and classical genetics has been developed over more than a century. The features include the possibility of wide interspeciﬁc crossing, biparental transmission of organelles, generation of fertile interspe- ciﬁc plastome–genome cybrids or hybrids, as well as relatively frequent reciprocal translocations of chromo- some arms that cause structural heterozygotic genomes. Collectively, these features allow the exchange of plastids and nuclei between species as well as the exchange of individual (or more) chromosome pairs or entire haploid chromosome sets. This often results in fertile but plastome–genome incompatible plants (Figure 1) and/or populations with varying chromosome formulas within species. Subsection Oenothera (=Euoenothera), the most intensely studied of the ﬁve subsections in the section Oenothera (5,22–24) is comprised of six basic nuclear genomes and ﬁve genetically and molecularly distinguish- able plastid types (I–V; 26–29) that are distributed as distinct nuclear genome combinations in approximately a dozen species, predominantly over the North American continent (22,23). To gain causal access to compartmental co-evolution of plant genomes and speciation processes, we have begun to develop cell and molecular biological approaches for the Onagracean genus. In preceding communications we have reported on the complete nucleotide sequence of the plastid chromosome of Oenothera elata subsp. hookeri strain johansen, representing the Oenothera plastome I (30), on an EST library derived from the Oenothera A genome (31), and on cell, tissue culture and transforma- tion protocols of that material (32–34). In this study we present and compare the complete nucleotide sequences of representatives of the ﬁve basic Oenothera plastomes and Nucleic Acids Research, 2008, Vol. 36, No. 7 2367 Figure 1. Plastome–genome compatibility/incompatibility in the sub- section Oenothera, redrawn with permission (5,26). A, B and C represent the basic nuclear genotypes, I–V the ﬁve genetically distinguishable plastomes. Genotypes boxed in red represent naturally occurring species. Minor symbols indicate variances noted for some nuclear subgenotypes. deduce their evolutionary relationship and temporal appearance. In a forthcoming study the phylogenetic relevance of the changes observed and potential plastome- encoded candidates responsible for interspeciﬁc plastome– genome incompatibility will be evaluated. MATERIALS AND METHODS Plastome sequencing Fully developed leaves of Oenothera elata subsp. hookeri strain johansen (plastome I; 35), Oe. biennis strain suaveolens Grado (plastome II; 36), Oe. glazioviana strain rr-lamarckiana Sweden (ex genetica: Oe. lamarckiana, platome III; 37), Oe. parviﬂora strain atrovirens (syn: Oe. cruciata, plastome IV; 38) and Oe. argillicola strain douthat 1 (plastome V; 39) were collected from 5- to 6-week-old greenhouse grown plants [for detailed taxon- omy of the subsection Oenothera see (23)]. DNA from plastome I was prepared from intact chloroplasts essen- tially as described in (40). Molecular cloning, sequencing and sequence analysis were performed as given in (11,30). Total DNA was isolated from plants carrying plastomes I to V, respectively, using the DNeasyTM Plant Mini Kit (Qiagen, Hilden, Germany). Since a number of errors were noted in the original sequence of plastome I (30), especially in the IR as also noted by others (41), its DNA was re-sequenced completely. PCR-derived fragments or frag- ments subcloned from plastid chromosomes serving as sequencing templates, spanning both DNA strands and overlapping a minimum of 200 bp, were generated over the entire chromosome of these plastomes. Almost the same, approximately 450, oligonucleotide primers (MWG Biotech, Ebersberg, Germany) employed for plastome I were used. Nucleotide sequences were determined by the dideoxy chain termination method (42) using energy- transfer ﬂuorochrome-labeled dideoxynucleotides (43) with an ABI 377 robot (Applied Biosystems, Darmstadt, Germany). Sequence data were ﬁrst subjected to the