The large inversion of 56 kbp 136 

phylogenetic marker, since it is shared among all Oenothera plastomes. It is absent in the closely related South American subsections Munzia and Raimannia (Hachtel et al., 1991) as well as in the sister group Epilobium (Schmitz and Kowallik, 1986). This observation and its almost identical endpoints in all five plastomes suggest that the inversion is not caused by one of the proposed rare parallel inversions (Downie and Palmer, 1994; Johansson, 1999; Tsumura et al., 2000). It has arisen monophyletically within the Oenothera clade and late in the history of the Onagracean complex, in the common ancestor of the subsection. Hence, the inversion marks a recent split in the history of the genus and predates the divergence of the subsection.

4.4.4. Patterns of subplastome variation in Oenothera populations

Other regions in the Oenothera plastomes reveal a higher phylogenetic resolution than the large inversion. The rrn16-trnIGAU spacer region, used as maker allele for the identification of

basic and subplatome types in crossing experiments (Chapters 3.1.1.3, 3.1.2.1 and 3.1.2.2), is also an indicator for gene flow und recent hybridization events within the subsection

Oenothera. The 17 alleles of the derived CAPS marker (Table 9) give an interesting spotlight on the phylogenetic relationship between plastomes and species. It could be shown that the region is suitable for such a phylogenetic analysis (Hornung et al., 1996; Sears et al., 1996). The marker is an indicator how much variation or subplastomes exist within the subgenus, without performing laborious RFLP analysis of the whole plastid chromosome (Herrmann et al., 1980). Nevertheless, the degree of variation is important to know, if differences between five sequenced reference plastomes, described this thesis, are going to be generalized in terms of speciation events.

The rrn16-trnIGAU allele provides a good confirmation of the monophyletic origin of plastome

IV in Oe. oakesiana (AC-IV) and Oe. parviflora (BC-IV). The current model claims that Oe. parviflora (BC-IV) arose as a hybrid between a hypothetical ancestor, with the genomic constitution CC-IV, and Oe. nutans (BB-III). Oe. parviflora (BC-IV) itself hybridized with

Oe. biennis (AB-II or BA-III) resulting in the AC-IV species Oe. oakesiana (Dietrich et al., 1997). Therefore, plastome IV of both species should be identical and monophyletic, which could be confirmed at the molecular level with the described marker allele IV1. All strains

tested for plastome IV had an identical sequence (Table 9), suggesting that plastome IV was already fixed in the population of the hypothetical ancestor CC-IV.

For plastomes of type I, the clearly distinguishable and specific alleles of Oe. elata subsp.

elata (AA-I1/2), Oe. elata subsp. hookeri (AA-I3, AA-I4) and Oe. villosa subsp. villosa (AA-I5)

(Table 9) indicate that gene flow between these three populations no longer occurs. This is likely due to their geographic distribution and propagation strategies (Dietrich et al., 1997). Although these species still carry genetically plastome I, the subplastomes seem to evolve in different directions. This is a clear example that Oenothera provides a suitable material not only to study speciation, but also pre-speciation processes.

Cleland (1962) mentioned that the border between plastome type II and III in Oenothera

strains, naturally occurring at the North American continent, is not sharp. The genetic behaviour of these plastomes in AB, BA and BB-species was somehow in between the plastomes of types II and III of Stubbe (1959). The distribution of rrn16-trnIGAU alleles in

different Oenothera strains of Oe. biennis (AB-II and BA-III), Oe. glazioviana (AB-III), Oe. grandiflora (BB-III) and Oe. nutans (BB-III) is consistent with the observation of Cleland (1962). Although for nearly all strains tested the genetic plastome type is known but no specific sequence for the rrn16-trnIGAU spacer region could be found for plastomes II or III

(Table 9). If the evolutionary history of the above mentioned species is considered, nearly all of them arose from hybridization events and there is more than one model present in the literature, how these species may have evolved (Cleland, 1972; Wasmund, 1990; Schumacher and Steiner, 1993; Dietrich et al., 1997). The data presented in this work indicate that there is still substantial gene flow within the genotypes under study, since alleles rrn16-trnIGAU II/III1

and II/III3 are present in more than one species. This is of particular interest, because the

plastome appears to play an inferior role in establishing hybridization barriers between Oe. grandiflora (BB-III), Oe. nutans (BB-III) and Oe. biennis (AB-II or BA-III), since the majority of all possible crosses between the three species result in compatible offspring (Figure 4). The data support the hypothesis that the plastome is the only strong hybridization barrier in the genus (Chapter 1.3), and if it is not very pronounced or missing, gene flow can occur in hybridization zones.

Detecting so many different rrn16-trnIGAU alleles of plastome II/III in the same species, but

also identical ones in different species (Table 9), suggests that plastomes II or III are not fixed so far in the different Oenothera populations. Consequently, their genetic behaviour is not

and delaware are associated with plastome II, but the biennis-2 strain micaville carries plastome III. Usually, strains of the microspecies biennis-1 and biennis-2 in Cleland’s taxonomy, are associated with plastomes III and II, respectively (Cleland, 1972). The data indicate that plastome types II and III in AB, BA or BB species still adapt to their nuclear background, offering the possibility to study very recent, ongoing evolutionary processes. In summary, plastomes in Oenothera are an excellent tool to monitor hybridization events and their impact for speciation. Functional genetic analysis of fast evolving parts of the Oenothera

plastome, related to physiological characterization of plastome-genome incompatibility in the genus should allow to draw a complete and definite picture of the evolution and mechanisms of speciation in the North American subsection Oenothera.

4.5. Selection pressure and determinants of PGI

What causes the differences between the Oenothera plastomes and what are the functional molecular determinants responsible for PGI? Incompatibility between nuclear and plastid genomes can lead to hybridization barriers of different strengths, with remarkable impact on speciation (Chapter 3.3.3), but which selection forces produce them?

4.5.1. Selection pressures acting on the plastome

That strong selection forces can act on the plastome is evident and well studied in Oenothera. In this thesis, bioinformatic comparisons of amino acid substitutions rates in the five basic

Oenothera plastomes uncovered remarkably higher mean Ka/Ks values, compared to averaged values found in angiosperms (Chapter 3.3.4). This indicates a substantial selection pressure on Oenothera plastome sequences. Similar results were recently obtained for clpP in

Silene and other genera (Erixon and Oxelman, 2008). Remarkably, the genus Silene also displays PGI phenotypes (Table 2).

The consequence of selection on a certain plastid type is chloroplast capture, the introgression of a plastid of one species into another one. This can happen relatively quickly, already if a small fitness advantage exists for the female (Tsitrone et al., 2003). The Triticeae tribe, with its members Triticum and Hordeum, represents a well studied example. In its interspecific hybrids strong preferences for distinct plastid genomes can be found (Redinbaugh et al.,

be monitored in nature, as in Oenothera, Silene or Triticae, and has been described by the theoretical models of chloroplast capture.

A major selection force acting on plastomes is obviously related to the photosynthetic process. It is one of the most important functions of the organelle and photosynthesis itself is the principal energy supplying reaction of a plant cell that influences important processes such as water balance and drought tolerance (see below). Indeed, differences in photosynthetic performance have been detected with different “cytoplasms” in introgression lines of Triticum and Aegilops (Iwanaga et al., 1978). A similar finding was reported from

Oenothera, in which different photosystem II yields in green plants could be genetically linked to two different compatible plastomes in the same nuclear background (Glick and Sears, 1994). Cold stress acting on photosynthesis could be excluded as a selection force (Dauborn and Brüggemann, 1996). Thus, although the coding potential of closely related plastomes is almost identical (Chapter 3.2), photosynthetic efficiency conveyed by closely related plastome types may differ, since the adaptation to their nuclear components is different. It is obvious, that these differences are result and subject of selection.

Regardless of whether the origin of genetic differences of photosynthesis traits is located in nuclear or in plastid genomes, these differences are found in natural populations and are influenced by selection. Higher photosynthetic rates are usually associated with growth and fitness advantages (Arntz and Delph, 2001). Selection often acts indirectly on photosynthetic traits via drought or oxidative stress. It can be monitored, e.g. by water-use efficiency or photosynthetic parameters (Arntz and Delph, 2001; Hura et al., 2007; Yang et al., 2007). The link of photosynthesis to drought stress consequently implies that periods of climate changes may introduce PGI and therefore built hybridization barriers resulting in speciation. It will be a fascinating future task to examine this hypothesis. For the genus Oenothera, drought stress is a very likely selection pressure for speciation, since appearance of the subsection

Oenothera was accompanied by a fluctuating climate for both, precipitation and temperature, during the Pleistocene (Cleland, 1972).

Another driving force could reside in the intrinsic structure of the plastid genome. Genes like

evolution rates in these regions could act on the DNA structure itself and perhaps provide reasons for PGI.