Polyploidy is a particularly important evolutionary mechanism in flowering plants [1–4]. During the past 70 years, many plant biologists have estimated the frequency of polyploidy in the angiosperms using analysis of base chromosome numbers [5–8], as well as measurements of stomatal size in fossil and extant taxa . Based on these approaches, researchers estimated that from 40% to 70% of angiosperms have experienced polyploidy in their evolutionary history [5–7, 9]. Recent genomic studies indicate, however, that polyploidy is even more prevalent in angiosperm lineages than previously suspected. Sequencing of the entire nuclear genome of Arabidopsis thaliana indicated two or three rounds of genome-wide duplication [10–17]. Complete genome sequences also indicate multiple ancient polyploidy events in Populus trichocarpa and Vitis vinifera [18–20]. Genomic data (including analyses of ESTs) indicate ancient polyploidy for other angiosperms , including the basal angiosperm Nuphar advena, the magnoliids Persea americana, Liriodendron tulipifera, and Saruma henryi, the basal monocot Acorus americanus, and the basal eudicot Eschscholzia californica . It now appears that all angiosperms may have undergone at least one round of genome duplication (reviewed in [23, 24]).
Several outcomes for duplicated genes are possible at the genomic and transcriptional levels. First, both members of a duplicate gene pair may retain their original function. Second, one copy of a duplicate gene pair may retain the original function, but the other copy may become lost or silenced [3, 13, 14, 23–26]. Third, duplicate genes may partition the original gene function (subfunctionalization), with one copy active, for example, in one tissue and the other copy active in another tissue [25, 27–31]. Fourth, one copy may retain the original function, while the other develops a new function (neofunctionalization) [32–38].
Recent studies have revealed varied consequences of genome evolution and gene expression following polyploidy in diverse angiosperms, including Arabidopsis [39–44] and crops such as cotton [31, 45, 46], wheat [1, 47–50], and Brassica [51–55]. Several investigations have shown that following polyploidy, rapid genomic rearrangement [48, 51, 56], gene loss [1, 49, 53], or gene silencing via DNA methylation [39, 41, 43, 44, 49, 53] may occur. However, few analyses have explored the genetic and genomic consequences of allopolyploidy in natural systems. Six natural allopolyploids are known to have formed within the past 150 years, thus affording the opportunity to examine the nearly immediate consequences of polyploidization in nature: Cardamine schulzii , Senecio cambrensis [58–63], Senecio eboracensis , Spartina anglica [64–68], and Tragopogon mirus and T. miscellus [26, 51, 69–72]. Several studies of these recently formed allopolyploids show evidence of either genomic or expression-level changes, relative to their diploid parents. For example, Salmon et al.  showed that methylation patterns differ between the hexaploid parents (Spartina maritima and S. alterniflora), the independently formed hybrids (Spartina × townsendii and S. × neyrautii), and the allopolyploid S. anglica (formed from Spartina × townsendii). In Senecio, hybridization of diploid S. squalidus with tetraploid S. vulgaris forms a sterile triploid, S. × baxteri, and subsequent genome duplication produced the allohexaploid S. cambrensis. Through microarray analysis of floral gene expression patterns in synthetic S. cambrensis lines, Hegarty et al. [62, 73] observed that the synthetic hybrid S. × baxteri showed immediate transcriptional changes compared to the parental expression patterns, and that this "transcriptional shock" was "subsequently calmed" in allohexaploid S. cambrensis, suggesting that hybridization and polyploidization have distinct effects on large-scale gene expression in this system.
One of the best systems for the study of naturally occurring polyploids is provided by the genus Tragopogon (Asteraceae). Tragopogon comprises ca. 100 to 150 species distributed throughout Europe, temperate Asia, and North Africa [74–76]. Three diploid species (T. dubius, T. porrifolius, and T. pratensis) were introduced from Europe into the Palouse region of eastern Washington and adjacent Idaho, USA, in the early 1900s [69, 70]. The introduction of these three diploid species brought them into close contact, and as a result, two allotetraploid species (T. mirus and T. miscellus) formed . First collected in 1949 , these recently formed polyploids are less than 80 years old. Morphological, cytological, flavonoid, isozymic, and DNA evidence confirmed the ancestries of these two allotetraploids [77–83]. Multiple lines of evidence suggest that T. miscellus has formed recurrently, possibly as many as 21 times, including reciprocal formation, and T. mirus has formed repeatedly perhaps 13 times (but not reciprocally) [70, 84, 85]. Therefore, T. mirus and T. miscellus afford unique opportunities for the investigation of recent and recurrent polyploid evolution. In fact, nearly every population of these species may have formed independently (V. Symonds et al., unpublished data).
Tate et al. [26, 86] and Buggs et al.  studied genomic changes and expression differences of homeologs within natural populations of Tragopogon miscellus, as well as in synthetic F1 hybrids and first-generation polyploids formed from the diploid parents T. dubius and T. pratensis. Most of the genes analyzed show additivity in T. miscellus at both the genomic (seven out of 23) and cDNA levels (12 out of 17). However, loss of one parental homeolog was observed at several loci (27 out of 46 homeologs), as were several examples of gene silencing (nine out of 34 homeologs). Both homeolog losses and silencing patterns vary among individuals in natural polyploid populations of independent origin [26, 87]. Changes were also detected in rDNA content  and expression  in populations of T. miscellus. Although T. miscellus has fewer rDNA repeats of T. dubius than of T. pratensis , apparently due to concerted evolution, most of the rDNA expression derives from the T. dubius repeats . The same pattern of rDNA expression has been observed in populations of T. mirus compared to its parents [71, 72]; T. mirus has fewer repeats of T. dubius than of T. porrifolius , but most of the rRNA is produced by the T. dubius copies . Although homeolog loss events and expression changes were observed in natural populations of T. miscellus, no such changes were observed in comparable analyses of F1 hybrids between the diploid parents, T. dubius and T. pratensis [26, 87], or in first-generation synthetic lines .
In this study we extend our examination of gene loss and differential expression to the polyploid T. mirus. In nature, T. mirus has formed repeatedly, but only when T. dubius is the paternal parent and T. porrifolius is the maternal parent [69, 82]. However, T. mirus can be produced synthetically in both directions with about equal frequency . Tragopogon mirus provides an opportunity to compare expression differences at the genomic and transcriptional levels with the results obtained for T. miscellus [26, 87]. Our main objectives were to: 1) investigate the genomic changes and expression differences of parental homeologs in T. mirus relative to its diploid parents, 2) determine the identity of the genes that exhibit those changes, and 3) assess whether individuals within and among recurrently formed natural populations of T. mirus show similar patterns of genome evolution and gene expression.