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The role of hybridization in evolutionary processes, including the generation of novel traits, introgression of traits between species, and even speciation, has received widespread attention (Stebbins, 1959; Arnold, 1992; Rieseberg & Carney, 1998; Abbott et al., 2009). In recent years it has become apparent that hybridization can lead to the generation of novel molecular and morphological phenotypes (Rieseberg et al., 2003; Kim et al., 2008). Such phenotypes can persist over evolutionary time and can even lead to speciation among hybrid lineages (Seehausen, 2004; Soltis & Soltis, 2009). At the metabolic level, hybridization can impact the diversity of secondary metabolites (SMs) in plants (Orians, 2000). SMs are important for mediating interactions between plants and their environment (Iriti & Faoro, 2009), and the composition of plant SMs can play a role in determining the evolutionary success of populations and species (e.g. Burow et al., 2010).
In the first (or F1) hybrid generation, most phytochemicals are expressed at concentrations that are either similar to those of one of the parents or intermediate between those of the two parents (Orians, 2000). However, recombination in F2 and later generation hybrids is expected to increase variation in phytochemical expression among different F2 genotypes. Transgressive segregation can occur, such that some F2 genotypes may vary outside the range observed in parental genotypes, and provide key variation upon which selection can act during the process of adaptation (Rieseberg et al., 1999, 2007). One of the drawbacks of many studies that quantify SM expression by hybrids is that only mean values are reported for each hybrid class (i.e. F1, F2, or backcross; e.g. Hallgren et al., 2003; O’Reilly-Wapstra et al., 2005). When genotypes are pooled within classes, transgressive phenotypes may not be identified. Also, many studies fail to carry out replicate measurements on genotypes within parental or hybrid classes, and such studies therefore fail to measure and test for genetically controlled variation in SM expression within these classes. In this study, we investigated variation among > 100 replicated F2 hybrids, which allowed us to conduct appropriate statistical testing to identify differences between and among hybrid and parental genotypes.
SM accumulation can be influenced by a number of factors, including genetics, abiotic factors (such as nutrient and light availability), biotic factors (including competition, herbivory and disease), and interactions between these factors (Lankau & Kliebenstein, 2009; Kirk et al., 2010). However, little is known about the mechanisms behind these complex regulatory systems. Recent work on the genomics and ecology of model and nonmodel species has started to shed light on the control of SM expression. For example, studies of glucosinolate expression in Arabidopsis thaliana have identified four major genetic loci responsible for the expression of 14 different glucosinolates (Kliebenstein, 2009). In addition to the regulatory complexity within individuals, there is considerable variation in SM profiles both within and among plant populations (e.g. Burow et al., 2010). Furthermore, more attention has been paid to SMs in above-ground plant parts than in below-ground plant parts, even though the latter are probably equally important to a species’ ecology, and there is often interaction or coordination between the expression of SMs in above-ground plant tissues and that in below-ground plant tissues (van Dam et al., 2009).
Species in the genus Jacobaea (syn. Senecio, Asteraceae) have been used to investigate the evolutionary basis of SM diversity in plants, because they contain a diverse but structurally related group of alkaloids that play a role in biotic interactions (e.g. Hartmann, 1999; Hol & van Veen, 2002; Macel & Vrieling, 2003; Macel et al., 2005; Kowalchuk et al., 2006). Twenty-six pyrrolizidine alkaloids (PAs) have been reported from 24 species of Senecio sect. Jacobaea (Pelser et al., 2005), although the recent development of more sensitive analytical methods has allowed the detection of a greater number of structural PA variants in the same species (Joosten et al., 2009, 2010, 2011). In Jacobaea species, all PAs except for senecivernine are derived from senecionine N-oxide. Senecionine N-oxide is synthesized in the roots, transported to the shoots via the phloem, and diversified into other PA structures (Hartmann & Toppel, 1987; Sander & Hartmann, 1989; Hartmann et al., 1989). Structurally derived PAs are thought to be produced from the precursor senecionine N-oxide via a limited number of steps (Hartmann & Dierich, 1998; see a schematic diagram representing putative PA biosynthetic pathways in Fig. S2). Apart from structural diversification, PAs do not undergo any turnover or degradation (Sander & Hartmann, 1989; Hartmann & Dierich, 1998). PAs can occur in plants in two forms: tertiary amine (free base) and N-oxide (Rizk, 1991; Wiedenfeld et al., 2008; Joosten et al., 2011). The proportion of tertiary amine is different among PAs and between genotypes. In Jacobaea plants, the tertiary amine form is usually present among higher proportions in jacobine-like PAs than among senecionine-like and erucifoline-like PAs. However, the mechanisms by which one form is converted to the other are not well understood (Joosten et al., 2011).
PA composition and concentration vary greatly among and within Jacobaea species (Witte et al., 1992; Macel et al., 2002, 2004; Pelser et al., 2005). Four different PA chemotypes of Jacobaea vulgaris are reported to occur; these include jacobine, erucifoline, mixed and senecionine chemotypes (Witte et al., 1992; Macel et al., 2004). Field studies and controlled bioassays that incorporate herbivores indicate that plant resistance to herbivorous invertebrates is correlated with plant PA concentration and composition (Leiss et al., 2009; Macel & Klinkhamer, 2010). Individual PAs have different deterrent effects on generalist herbivores (Macel et al., 2005), and also have different stimulatory effects on the oviposition of the specialist herbivore Tyria jacobaeae (the cinnabar moth; Macel & Vrieling, 2003). Furthermore, free base PAs appear to have different effects on generalist herbivores compared with their corresponding N-oxides (van Dam et al., 1995; Macel et al., 2005). These cumulative findings indicate that PA diversity is ecologically important with respect to interactions between plants and herbivores.
Interspecific hybridization is widespread in the Senecio genus, including section Jacobaea (e.g. Vincent, 1996). For example, hybridization between Senecio squalidus and Senecio vulgaris led to the origin of three new fertile hybrid taxa, and S. squalidus itself is a hybrid species resulting from a cross between Senecio aethnensis and Senecio chrysanthemifolius (Abbott & Lowe, 2004; James & Abbott, 2005; Abbott et al., 2009). There are many other well-documented cases of hybridization between Senecio species (e.g. Beck et al., 1992; Hodalova, 2002; Lopez et al., 2008), including natural hybridization between J. vulgaris (formerly Senecio jacobaea L.) and J. aquatica (formerly Senecio aquaticus L.) which occurs in The Zwanenwater Nature Reserve in the Netherlands (Kirk et al., 2004).
Jacobaea vulgaris (tansy ragwort or common ragwort) is native to Europe and west Asia but is invasive in North America, Australia and New Zealand. Jacobaea aquatica (marsh ragwort) is closely related to, but not a sister species of, J. vulgaris (Pelser et al., 2003). The two species are ecologically distinct. Jacobaea vulgaris often occurs in dry, sandy soil with little organic matter and J. aquatica is found in wet habitats in soils that are high in organic matter. The two species are attacked by different guilds of herbivorous insects in the field. Different susceptibility to a generalist herbivore has been observed (Kirk et al., 2004, 2010). Putative hybrids from the Zwanenwater (the Netherlands), initially identified in 1979 based on highly variable and usually intermediate flower and leaf lobe morphology compared with J. vulgaris and J. aquatica, were confirmed to be hybrids between these two species using molecular genetic markers and PA composition (Kirk et al., 2004). The natural hybrid population is highly backcrossed with J. vulgaris, and F1 hybrids are uncommon in the natural population (Kirk et al., 2004, 2005). In contrast to J. vulgaris, J. aquatica lacks jacobine-like PAs but is rich in senecionine-like PAs (Kirk et al., 2010). A previous study that characterized PA composition of natural hybrids and artificial F1 hybrids of the two species showed that PA expression was affected by species and environment interactions (Kirk et al., 2010).
To obtain a hybrid family, we selected a J. vulgaris genotype of the jacobine chemotype, which is rich in jacobine-like PAs, and a J. aquatica genotype. We established an artificial J. vulgaris × J. aquatic family, which includes two parental genotypes, two F1 hybrids, and c. 100 different F2 hybrid genotypes. These are all kept in tissue culture and can be reproduced at length. The hybrid system to a great extent overcomes the problem of unavailability of the relevant pure PAs for the study of the effects of individual alkaloids or PA combinations. Joosten et al. (2011) demonstrated the genetically controlled presence of tertiary PAs in Jacobaea species using this system. Kirk et al. (2011) reported transgressive segregation of primary and secondary metabolites in the F2 hybrids of this cross using NMR-based metabolomics.
In this study, we aimed to investigate whether hybridization can generate new PA variation in this system and to gain an initial understanding of how PA accumulation is genetically regulated based on the pattern of PA variation. We focused on differences in PA expression among segregating hybrids originating from a single cross between two parental genotypes, and we grew plants under standard conditions to eliminate the effect of environment on PA expression. The methods used in this study differed from those used in previous work in two respects: first, the large numbers of genotypes and replications resulted in a very large sample size; second, we measured PAs by LC-MS/MS, which is highly sensitive and can detect the two forms of PAs simultaneously (Joosten et al., 2011). We addressed the following questions: Do F2 hybrids produce novel PAs? Does any F2 hybrid genotype show evidence of transgressive variation (over-expression or under-expression) with regard to the concentrations of total PA, a structural group of PAs, or any individual PAs? Does hybridization produce novel PA compositions among F2 genotypes? Is there covariation in the expression of individual PAs? Are there correlations between the accumulation of PAs in the roots and that in the shoots?
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Fig. S1 Structures of the pyrrolizidine alkaloids (PAs) found in shoots and roots of Jacobaea aquatica, Jacobaea vulgaris, and F1 and F2 hybrids.
Fig. S2 Putative biosynthetic pathways for diversification of pyrrolizidine alkaloids (PAs) in the Jacobaea section.
Fig. S3 Frequency distribution of genotypic mean concentrations of pyrrolizidine alkaloids (PAs) from four structural groups in the shoots and roots of 102 F2 hybrid genotypes between Jacobaea aquatica and Jacobaea vulgaris.
Fig. S4 Pyrrolizidine alkaloid (PA) composition in the shoots and roots plotted by two-dimension nonparametric multidimensional scaling (NMDS) and the loadings.
Table S1 Pyrrolizidine alkaloid (PA) concentrations in the shoots of Jacobaea aquatic (one genotype), Jacobaea vulgaris (one genotype), F1 hybrids (two genotypes) and F2 hybrids (102 genotypes)
Table S2 Pyrrolizidine alkaloid (PA) concentrations in the roots of Jacobaea aquatic (one genotype), Jacobaea vulgaris (one genotype), F1 hybrids (two genotypes) and F2 hybrids (102 genotypes)
Table S3 Quantitative variation of pyrrolizidine alkaloids (PAs) in the shoots and roots of two F1 and 102 F2 hybrids relative to parental genotypes (one genotype each of Jacobaea aquatica and Jacobaea vulgaris)
Table S4 Coefficients (rs) of Spearman rank correlation between the individual pyrrolizidine alkaloid (PA) concentrations in the shoots and roots of Jacobaea aquatic (one genotype), Jacobaea vulgaris (one genotype), F1 hybrids (two genotypes) and F2 hybrids (102 genotypes)
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