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- Materials and Methods
- Supporting Information
Interspecific hybridization is considered to be a process with important evolutionary consequences, both in plants and in animals (Stebbins, 1950; Anderson & Stebbins, 1954; Arnold, 1992, 1997; Dowling & Secor, 1997; Rieseberg, 1997; Seehausen, 2004; see also Schemske, 2000). A first and crucial step in the process involves the creation of F1 hybrids. Their viability, fertility and ecological position combined with the availability of a suitable habitat determine whether hybridization is incidental and without further consequences, or leads to further hybrid or backcross generations. These could eventually form a new evolutionary lineage (hybrid speciation; see Arnold, 1992, 1997; Rieseberg, 1997), or a permanent hybrid swarm maintained by repeated backcrossing (Barton & Hewitt, 1985). Such backcrossing may result in the introgression of genes (Anderson & Hubricht, 1938; Anderson, 1949; Stebbins, 1950; Rieseberg & Wendel, 1993; Arnold, 2004) giving rise to new allelic combinations and novel phenotypes (Arnold, 1992, 1997; Rieseberg & Carney, 1998; Rieseberg et al., 2000).
Hybrid swarms are often found in disturbed habitats (Anderson, 1948; Stebbins, 1950; Anderson & Stebbins, 1954). Flooding disturbance has played an important role in the formation and maintenance of hybrid swarms, for example involving Iris fulva and Iris brevicaulis (e.g. Johnston et al., 2004; Martin et al., 2006), Jacobaea vulgaris (formerly Senecio jacobaea) and Senecio aquatica (Kirk et al., 2005) and the Rorippa × anceps hybrid complex (Jonsell, 1968; Bleeker, 2007). In the latter system, the parental species are the self-incompatible perennials Rorippa amphibia and Rorippa sylvestris, both common along all major rivers in Europe. The distributions of the species within the floodplain suggest that they have become adapted to microhabitats with different flooding regimes (Blom, 1999). Rorippa amphibia occurs in sites with relatively stable water tables, mostly as emergent plants, or in dense, constantly moist grassland vegetation. Rorippa sylvestris prefers more open, ephemeral sites on riverbanks that can be flooded for longer periods, but can also dry out completely in summer (Jonsell, 1968; Blom, 1999).
Rorippa amphibia (mainly diploid and tetraploid) and R. sylvestris (mainly tetraploid and hexaploid) often grow in sympatry, presumably because sedimentation and erosion shape river floodplains into a mosaic of habitats. The species are interfertile at the tetraploid level and the hybrid Rorippa × anceps can easily be generated and backcrossed with both parents in the glasshouse (Jonsell, 1968; Bleeker, 2004; Stift, 2007). At several locations along the rivers Elbe (Germany) and Wisla (Poland), the presence of a range of intermediate morphologies suggests that hybridization and backcrossing have led to the formation of hybrid swarms (Bleeker, 2007). Molecular analyses of amplified fragment length polymorphism (AFLP) and chloroplast trnL/F intergenic spacer DNA sequences (Bleeker & Hurka, 2001; Bleeker & Matthies, 2005) and patterns of microsatellite variation of both parental species and putative hybrids (E. H. McLean, unpublished data) have confirmed these morphological indications of introgressive hybridization along the Elbe river. The availability of hybrids and the position of Rorippa within the tribe Cardamineae (Al-Shehbaz et al., 2006), which belongs to the same phylogenetic lineage (I) as the genomic model species Arabidopsis thaliana (Beilstein et al., 2006), provide excellent opportunities for comparative genomics (Schranz et al., 2007). Thus, Rorippa may function as a model system with which to unravel the genetic basis of traits that are associated with flooding tolerance.
The first aim of this research was to quantify the reactions of R. amphibia and R. sylvestris in coping with different flooding regimes. We measured growth, biomass allocation, dry:fresh weight ratios and leaf morphological traits under well-drained, waterlogged and fully submerged conditions. In concordance with its stable wet to waterlogged habitat, we predicted that R. amphibia would grow well in both drained and waterlogged treatments, but that R. sylvestris would exhibit the most vigorous growth under well-drained conditions and a growth reduction under waterlogged conditions. We expected submergence to reduce growth in both species and to affect leaf morphology and positioning (Voesenek et al., 2006). Moreover, we hypothesized that R. amphibia shoots would grow more when submerged in an attempt to reach the surface, thus possibly depleting available carbohydrate resources (Groeneveld & Voesenek, 2003); such a strategy would be advantageous in a habitat where flooding tends to be shallow and predictable. By contrast, we expected that R. sylvestris would arrest growth (thus storing carbohydrate reserves) and passively wait for better times, perhaps in a state of anaerobic dormancy (Laan & Blom, 1990; Vartapetian & Jackson, 1997). This would be advantageous in a habitat where flooding is deep and less predictable in duration. We also expected that R. sylvestris would generally allocate more biomass to root and rhizome storage tissue, to fuel its more vigorous regeneration from rhizomes (Jonsell, 1968).
The second aim of this research was to assess how the parental traits associated with the flooding regimes are expressed in first-generation (F1) hybrids obtained from glasshouse crosses between wild-collected plants of the two species. We asked whether their phenotypic expression is conducive for ecological divergence. More specifically, we tested whether F1 hybrids are intermediate with respect to the parental species (i.e. whether the expression of parental traits showed ‘genomic additivity’), mostly resemble one of the parents (‘genomic dominance’), or have trait values beyond those of either parent (‘genomic overdominance’).
Knowledge of how putatively adaptive traits (in this case associated with the different habitat preferences of R. amphibia and R. sylvestris) are expressed in F1 hybrids will contribute to answering broader evolutionary questions regarding the long-term consequences of hybridization. Are hybrids merely present because of their constant formation in disturbed habitats (Schemske, 2000), or do they have the potential to occupy a specific novel niche (Arnold, 1997)? Do the ecological characteristics of first-generation hybrids promote reproductive isolation, which might eventually lead to a separate hybrid lineage or even a new species (Rieseberg, 1997; Buerkle et al., 2000)? Or is it more likely that they will set the stage for backcrossing (to one or both parental species), making introgression and gene exchange the most important consequences of hybridization (Anderson & Hubricht, 1938; Whitney et al., 2006; Gross et al., 2007)?
- Top of page
- Materials and Methods
- Supporting Information
The first aim of this paper was to compare R. amphibia (denoted ‘A’ in figures and tables) and R. sylvestris (denoted ‘S’ in figures and tables) in terms of their responses to different flooding regimes. The former species occurs in more stable, constantly wet to waterlogged habitats, and the latter in habitats with a more unpredictable regime of intermittent (often prolonged and deep) flooding and drought episodes. We set out with a specific set of expectations of species-specific responses to three treatments, chosen to be representative of the naturally prevailing water regimes (except drought). Our data indeed revealed pronounced differences between the species.
Our results support the hypothesis that R. amphibia is better able to cope with waterlogging than R. sylvestris. In contrast to R. sylvestris, R. amphibia growth hardly differed between the well-drained and waterlogged conditions. Leaf formation, morphology and positioning were not much affected by waterlogging either. This apparent waterlogging tolerance may be related to the ability of R. amphibia to develop adventitious and aboveground roots in the water surface layer, which is a typical response for plants of waterlogged environments (Armstrong et al., 1994; Blom & Voesenek, 1996; Vartapetian & Jackson, 1997). In Rumex spp. similar species differentiation was found in the ability to form adventitious roots (Visser et al., 1996). Rorippa amphibia also showed an unexpected response. The leaf size of the longest leaf of R. amphibia increased significantly upon waterlogging, an effect not seen in R. sylvestris. Future studies should provide insight into the physiological basis of this leaf size difference, and its potential adaptive significance.
According to expectations, R. sylvestris showed the most vigorous growth under well-drained conditions, and high allocation to root growth. This corresponds to the hypothesis that under favourable conditions this species builds up carbohydrate reserves that are stored in the roots (Mooney, 1972), thereby increasing its regeneration capacity after adverse conditions (e.g. waterlogging or submergence).
The effect of submergence was comparable for the two parental species. Contrary to our expectation that R. sylvestris would arrest growth when submerged, both R. amphibia and R. sylvestris established significant growth over 2 wk under submerged conditions. Growth was reduced compared with the well-drained and waterlogging treatments and restricted to the shoot. In R. amphibia shoot growth was associated with a loss of root biomass. This suggests that submerged R. amphibia plants cannot photosynthesize underwater at rates that are sufficient to provide the carbon needed for the observed shoot growth. Possibly, they deplete their carbohydrate reserves in the taproot in an attempt to reach the surface. Mobilization of starch reserves upon submergence has been previously reported in rice (Oryza sativa; Raskin & Kende, 1984) and in Rumex palustris (Groeneveld & Voesenek, 2003). This will be advantageous if flooding is shallow so that restoring air contact is indeed possible. Alternatively, root growth in R. amphibia may be hampered as a consequence of a limited diffusion of oxygen to the roots, as a result of lower porosities or substantial radial oxygen loss (Visser et al., 2000). In contrast to R. amphibia, the shoot growth in R. sylvestris was not accompanied by a reduction of root biomass. This suggests that R. sylvestris can survive (and even continue to grow) under submerged conditions without depleting its reserves. It appears that R. sylvestris may be capable of underwater photosynthesis (Mommer & Visser, 2005) at a rate sufficient to support overall growth. Moreover (in contrast to R. amphibia), the leaf petiole of R. sylvestris elongated and the orientation of the youngest leaf became almost vertical, suggesting a hyponastic response (Voesenek et al., 2006). Clearly, R. sylvestris does not passively wait for better times in a state of complete anaerobic dormancy (Laan & Blom, 1990; Vartapetian & Jackson, 1997), at least not under the (light) conditions in our experiment. In Rumex species, survival under submerged conditions was reduced in the absence of light (Nabben et al., 1999). It was shown that shading may have an additional effect on growth in several waterlogging-tolerant species (Lenssen et al., 2003). The apparent tolerance to both waterlogging and submergence of the Rorippa species calls for future experiments to test the effect of shade in this system. In addition to this, (micro)habitat differences between R. amphibia and R. sylvestris could be explained by soil characteristics and differences in drought tolerance or drought avoidance strategies (Lenssen et al., 2005; Touchette, 2007). Although this has not been addressed in the current study, preliminary results of ongoing work indicate that R. sylvestris is capable of surviving longer periods of drought than R. amphibia, which may explain its occurrence in more drought-prone habitats (dykes, agricultural fields and roadsides) that are not necessarily affected by flooding.
Our second aim was to assess how parental traits associated with flooding are expressed in first-generation (F1) hybrids. For each trait, we explicitly tested whether the average of the hybrids deviated from the hypothesis of intermediacy. If it did so, we subsequently tested for dominance of either parent and overdominance. In a study of gene expression in a maize (Zea mays) F1 hybrid, additivity was found to be the most common mode of gene action between (inbred) parental lines (Swanson-Wagner et al., 2006). We could not reject intermediacy for the majority of cases, which may indicate that the rule of additivity also applies if the hybridizing parents are outbred. However, for a number of traits we rejected the intermediacy hypothesis and observed dominance of parental species traits. This may be explained in several ways. First, the parental species may have fixed genetic differences for a few genes with a major effect on growth and nonadditive gene action, similar to the situation of hybridizing two inbred parents (Swanson-Wagner et al., 2006). Secondly, many individual genetic effects may be channelled to a common metabolic pathway which results in nonadditivity of the phenotypic expression. Finally, the observed patterns may be explained by nucleolar dominance, a phenomenon where the genome of one parent is silenced (see Pikaard (2000) for a review). Note also that tetraploid F1 hybrids consist of two complete sets of genes, one from each parent. Epistatic interactions in F1s are therefore presumably different from those in later generations, where some parental genes could be absent, in particular as the mode of inheritance in the hybrid tetraploids is not disomic (Stift et al., 2008). Current research is comparing the expression profiles of the parental species with those of F1 and further hybrid (backcross) generations. From an ecological perspective, nonadditive expression of traits may have consequences for the habitat preference of hybrids. If hybrids resemble one of the parents (dominance), the hybrid habitat may overlap with that of the parent it resembles (Anderson, 1948). In particular, the typical R. amphibia waterlogging responses were dominantly expressed in hybrids. As in R. amphibia, waterlogging did not have an effect on growth and triggered the formation of adventitious roots, aboveground roots and a longer longest leaf. When submerged, hybrids also showed a root biomass reduction, indicating that (like R. amphibia) hybrids are perhaps less efficient in terms of underwater photosynthesis compared with R. sylvestris. If so, the hybrid habitat may thus mostly overlap with that of R. amphibia in locations where the occurrence of the parental species is mainly determined by flooding. In such a setting, introgression and (further) backcrossing would be more likely to happen in the direction of R. amphibia (Anderson & Hubricht, 1938).
In summary, our results supported our hypothesis that R. amphibia is better able to cope with waterlogging than R. sylvestris and provide insight into the traits that underlie the specialization of R. amphibia to waterlogged habitats. Additionally, we have shown that shoot growth under submerged conditions caused root biomass loss in R. amphibia, whereas R. sylvestris could prevent such loss. Furthermore, we have shown that R. amphibia leaf morphology remained constant, while R. sylvestris leaf morphology changed markedly upon submergence. Taken together, our results indicate that the two species are clearly different in their ways of coping with flooding. We are currently further unravelling the mechanisms that underlie these differences, making use of the extensive physiological knowledge of flooding tolerance in Rumex spp. (Voesenek et al., 2006), the genomic resources available for A. thaliana and the hybrids in our system. We found that hybrids combine a complex suite of traits from both parents, sometimes determined by additive, sometimes by dominant and rarely by overdominant expression of the parental phenotypes. This means that Rorippa × anceps F1 hybrids possess a unique phenotype, consisting of a combination of traits of both parental species. This specific hybrid phenotype may facilitate establishment of hybrids in free meandering rivers, particularly in the floodplains of rivers such as (amongst others) the Elbe and Wisla. Glasshouse crosses have corroborated field observations that hybrids are fertile and backcross readily to both parental species (Stift, 2007), making introgressive hybridization (Anderson & Hubricht, 1938) the most likely evolutionary consequence of hybridization between R. amphibia and R. sylvestris. Neutral microsatellite markers have revealed an interesting mixture of disomic and tetrasomic inheritance in F1 hybrids (Stift et al., 2008), which raises questions concerning the extent of the potential for transgressive segregation in further hybrid generations (backcrosses). We are currently investigating these further generations under both natural and glasshouse conditions, at the phenotypic, genetic and transcriptional levels.