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Nuclear DNA content in angiosperms (2C-value) ranges from 0.431 pg for Arabidopsis (Schmuths et al., 2004) to 254.8 pg for Fritillaria assyriaca (Bennett & Leitch 2003). This range in C-value is not entirely dependent on changes in ploidy, nor is it correlated with organismal complexity. For example, within diploid species in Poaceae, genome sizes range from 1.11 pg in Oryza sativum to 20.90 pg in Secale cereale (Bennett & Leitch 2003), with no apparent differences in complexity between these taxa. This variation in nuclear DNA content and its lack of correlation with gene number has been termed the ‘C-value paradox’ (Thomas, 1971) or ‘C-value enigma’ (Gregory, 2001).
Adaptive explanations have been sought for differences in genome size. Differences in genome size are correlated with slope aspect in Ceratonia siliqua (Bures et al., 2004), with frost resistance in the British flora (MacGillivray & Grime, 1995), with calyx size in Silene (MeaGher & Costich, 1996) and with elevation in Patagonian species of Berberis (Bottini et al., 2000) and European species of Dactylis (Reeves et al., 1998). Studies on invasive pines have found that smaller genomes are correlated with smaller seed size and higher invasiveness (Rejmanek & Richardson, 1996; Grotkopp et al., 2004). Alternatively, nonadaptive explanations have posited that increases in genome size are the result of deleterious mutations which fix via drift in small populations (Lynch & Conery, 2003). The observation that rare and endangered species, which usually have reduced population sizes, have larger genomes than more common species within their families (Vinogradov, 2003) is in accord with this proposal.
In the past decade, research has shifted to understanding the mechanisms behind genome size changes in plants. Polyploidy has been seen as a major source of increasing genome size. However, studies of natural polyploids within Asteraceae have shown that, while total DNA content increases on average with increasing ploidy level, the DNA content of each genome (i.e. the G1 nuclear DNA content divided by the ploidy level) in the polyploid nucleus decreases (Leitch & Bennett, 2004). Differences in nuclear DNA content within diploid plants have been linked to differences in intron size (Petrov, 2001) and transposon copy number (Bennetzen, 2002). Decreases in genome size correlate with a mutational bias towards deletions over insertions (Petrov, 2001), and illegitimate recombination has been shown to eliminate retrotransposon sequences (Bennetzen, 2002; Devos et al., 2002; Ma et al., 2004).
Newly synthesized plant polyploids may undergo extensive genomic changes. Hybridization followed by chromosome doubling leads to loss of DNA sequences in Brassica and Aegilops (Song et al., 1995; Liu et al., 1998a,b; Ozkan et al., 2001). In addition, some polyploid lineages exhibit increased retrotransposon activity (Ozkan et al., 2001). However, polyploidization does not automatically entail genomic restructuring: studies of resynthesized cotton polyploids (Gossypium; Liu et al., 2001) and recently derived natural polyploids (Tragopogon mirus and Spartina anglica) failed to find genomic changes (Baumel et al., 2002; Soltis et al., 2004).
Several studies have explored the timing of genomic change in polyploid lineages, and it now appears that some genomic changes are initiated in first-generation diploid hybrids, whereas others are exclusive to polyploidization (e.g. Song et al., 1995; Ozkan et al., 2001; Osborn et al., 2003). In Aegilops, for example, elimination of sequences unique to one of the parental genomes but found on multiple chromosomes begins in F1 plants and is completed in just two or three generations after polyploidization (Ozkan et al., 2001; Shaked et al., 2001). In contrast, sequences unique to a single chromosome from one parental genome are maintained in diploid hybrids, but are rapidly lost following polyploidization (Ozkan et al., 2001). Similar patterns have been reported from studies of diploid hybrids. Some plant and animal hybrids show genomic changes upon hybridization, including fruit flies, wallabies, and beans (Rogers & Bendich, 1987; Petrov et al., 1995; O’Neill et al., 1998; Labrador et al., 1999), whereas others are entirely stable (Guerreiro, 1996).
Despite substantial research on the genomic consequences of hybridization and polyploid speciation, genome size changes in diploid or homoploid hybrid species remain to be explored. Sims & Price (1985) reported nuclear DNA contents for 19 diploid sunflower (Helianthus) species, and it was later shown that three of these (Helianthus anomalus, Helianthus deserticola, and Helianthus paradoxus) are diploid hybrid derivatives of the same parents, Helianthus annuus and Helianthus petiolaris (Rieseberg, 1991). Intriguingly, the three homoploid hybrid species were reported to have substantially more DNA than their parents. However, only three individuals were analyzed in each species. Also, discovery of the influence of plant secondary compounds on the estimation of DNA content by both Fuelgen densitometry and flow cytometry (Greilhuber, 1988; Price et al., 2000) has led to changes in practice and created uncertainty regarding many earlier reported C-values.
Hybrid sunflowers offer several advantages for the study of genome-size evolution in homoploid hybrid species and its potential adaptive consequences. First, two of the three species (H. anomalus and H. deserticola) appear to have arisen multiple times in nature (Schwarzbach & Rieseberg, 2002; Gross et al., 2003), so we can ascertain whether the same genomic changes have occurred independently in the wild. Secondly, numerous hybrid zones between the parental species exist naturally. These zones can be exploited to ask whether the genome size variation found in the ancient hybrid species occurs in natural hybrid zones. Finally, genetic mapping studies indicate that the genomes of the hybrid species were extensively restructured during the speciation process (Rieseberg et al., 1995, 1996, 2003). Glasshouse experiments have shown that the chromosomal changes that separate the hybrid species from the parents can be largely duplicated after just four generations of fertility selection (Rieseberg et al., 1996; Rieseberg, 2000). Thus, we can use this unique germplasm to ask whether genome size variation can be replicated in the glasshouse and whether the ‘genomic shock’ caused by hybridization can generate DNA content variation.
In this study, we examined multiple individuals from multiple populations of the three homoploid hybrid sunflower species and the two parents to confirm previous reports of a DNA-content shift and to assess variation among independently derived populations. We also examined individuals from natural hybrid zones, synthetic F1 individuals, and synthetic F6 individuals in order to understand the timing of changes in genome size in the speciation process.