By comparing the expected genome size of the polyploid (based on summing the genome size of species identified as either a parent or most closely related to the diploid progenitors) with the observed genome size, four polyploids showed genome downsizing and five showed increases. There was no discernable pattern in the direction of genome size change with age of polyploids, although with increasing age the amount of genome size change increased. In older polyploids (approx. 4.5 million years old) the increase in genome size was associated with loss of detectable genomic in situ hybridization signal, whereas some hybridization signal was still detected in species exhibiting genome downsizing. The possible significance of these results is discussed.

Summary• Here, we analyze long-term evolution in Nicotiana allopolyploid section Repandae (the closest living diploids are N. sylvestris , the maternal parent, and N. obtusifolia , the paternal parent). We compare data with other more recently formed Nicotiana allopolyploids.• We investigated 35S and 5S nuclear ribosomal DNA (rDNA) chromosomal location and unit divergence. A molecular clock was applied to the Nicotiana phylogenetic tree to determine allopolyploid ages.• N. tabacum and species of Repandae were c . 0.2 and 4.5 Myr old, respectively. In all Repandae species, the numbers of both 35S and 5S rDNA loci were less than the sum of those of the diploid progenitors. Trees based on 5S rDNA spacer sequences indicated units of only the paternal parent.• In recent Nicotiana allopolyploids, the numbers of rDNA loci equal the sum of those of their progenitors. In the Repandae genomes, diploidization is associated with locus loss. Sequence analysis indicates that 35S and 5S units most closely resemble maternal and paternal progenitors, respectively. In Nicotiana , 4.5 Myr of allopolyploid evolution renders genomic in situ hybridization (GISH) unsuitable for the complete resolution of parental genomes.

BackgroundPolyploidy, frequently termed “whole genome duplication”, is a major force in the evolution of many eukaryotes. Indeed, most angiosperm species have undergone at least one round of polyploidy in their evolutionary history. Despite enormous progress in our understanding of many aspects of polyploidy, we essentially have no information about the role of chromosome divergence in the establishment of young polyploid populations. Here we investigate synthetic lines and natural populations of two recently and recurrently formed allotetraploids Tragopogon mirus and T. miscellus (formed within the past 80 years) to assess the role of aberrant meiosis in generating chromosomal/genomic diversity. That diversity is likely important in the formation, establishment and survival of polyploid populations and species.Methodology/Principal FindingsApplications of fluorescence in situ hybridisation (FISH) to natural populations of T. mirus and T. miscellus suggest that chromosomal rearrangements and other chromosomal changes are common in both allotetraploids. We detected extensive chromosomal polymorphism between individuals and populations, including (i) plants monosomic and trisomic for particular chromosomes (perhaps indicating compensatory trisomy), (ii) intergenomic translocations and (iii) variable sizes and expression patterns of individual ribosomal DNA (rDNA) loci. We even observed karyotypic variation among sibling plants. Significantly, translocations, chromosome loss, and meiotic irregularities, including quadrivalent formation, were observed in synthetic (S0 and S1 generations) polyploid lines. Our results not only provide a mechanism for chromosomal variation in natural populations, but also indicate that chromosomal changes occur rapidly following polyploidisation.Conclusions/SignificanceThese data shed new light on previous analyses of genome and transcriptome structures in de novo and establishing polyploid species. Crucially our results highlight the necessity of studying karyotypes in young (<150 years old) polyploid species and synthetic polyploids that resemble natural species. The data also provide insight into the mechanisms that perturb inheritance patterns of genetic markers in synthetic polyploids and populations of young natural polyploid species.

We investigated concerted evolution of rRNA genes in multiple populations of Tragopogon mirus and T. miscellus, two allotetraploids that formed recurrently within the last 80 years following the introduction of three diploids (T. dubius, T. pratensis, and T. porrifolius) from Europe to North America. Using the earliest herbarium specimens of the allotetraploids (1949 and 1953) to represent the genomic condition near the time of polyploidization, we found that the parental rDNA repeats were inherited in roughly equal numbers. In contrast, in most present-day populations of both tetraploids, the rDNA of T. dubius origin is reduced and may occupy as little as 5% of total rDNA in some individuals. However, in two populations of T. mirus the repeats of T. dubius origin outnumber the repeats of the second diploid parent (T. porrifolius), indicating bidirectional concerted evolution within a single species. In plants of T. miscellus having a low rDNA contribution from T. dubius, the rDNA of T. dubius was nonetheless expressed. We have apparently caught homogenization of rDNA repeats (concerted evolution) in the act, although it has not proceeded to completion in any allopolyploid population yet examined. (Ownbey 1950). The introduction of these dipsequences frequently show evidence of homogenizaloid species into the Palouse brought them into close tion, probably caused by processes such as unequal contact, something that rarely occurs in the Old World crossing over and gene conversion, mechanisms collecwhere the diploids are largely allopatric. Using mortively referred to as concerted evolution (Zimmer et al. phology and cytology, Ownbey (1950) demonstrated 1980;Dover 1982). The process of concerted evolution that T. mirus and T. miscellus are allotetraploids (2n ϭ can best be studied in synthetic polyploids or in natural 24) whose diploid (2n ϭ 12) parents are T. dubius and polyploids of clear and very recent ancestry. However, T. porrifolius and T. dubius and T. pratensis, respectively. only a few naturally occurring polyploid species areThe ancestries of both tetraploids were subsequently known to have arisen spontaneously within the past 150 confirmed through flavonoid, isozymic, and DNA studyears: Cardamine schulzii (Urbanska et al. 1997) clones from the polyploids suggested that the parental and P. S. Soltis, personal observation).rDNA types were not present in the allotetraploids in Variable numbers of rRNA genes coding for 18S-5.8S-equal frequency. In fact, T. dubius appeared to be under-26S RNA occur in different plant species (between 1000 represented in both allotetraploid species. To assess and Ͼ50,000 genes), forming multigene families in long whether concerted evolution has been operating in the tandem arrays (Hemleben and Zentgraf 1994). The recently formed allotetraploids, T. mirus and T. miscellus, intragenic (ITS) and intergenic spacers (IGS) associwe investigated the rDNA cistron using cloning, Southated with genic regions often vary among species and ern blots, slot blots, and single-str...

The nuclear cytoplasmic interaction (NCI) hypothesis of genome evolution and speciation in plants states that newly formed allopolyploids pass through a bottleneck of sterility and the fertile plants that emerge are fixed for speciesspecific chromosome translocations. These translocations restore fertility and reduce negative effects of the maternal cytoplasm on an alien paternal genome. Using fluorescent in situ hybridization and genomic in situ hybridization and by reviewing published data, we test the NCI hypothesis using three natural Nicotiana allotetraploids (all 2 n = 4 x = 48, N. arentsii , N. rustica and several genotypes, including a feral plant and cultivars, of N. tabacum (tobacco)). We compare these data with three synthetic tobacco plants (Th37) that are F3 descendent progeny of an allotetraploid formed from Ǩ N. sylvestris (2 n = 24) ¥ ǩ N. tomentosiformis (2 n = 24). No intergenomic translocations were observed in N. arentsii and N. rustica . An analysis of subtelomeric tandem repeats in these allotetraploids and their putative parents shows minimal genetic changes; those that do occur may reflect evolution in the diploids or the polyploids subsequent to allopolyploidy. All natural N. tabacum genotypes have intergenomic translocations. This may reflect a large 'genomic-shock' generated by allopolyploidy involving widely diverged parental species. Two of three synthetic tobacco plants had a translocation similar to that found in all cultivars of tobacco. This translocation may be significant in tobacco fertility and may have been fixed early in tobacco's evolution. But it is lacking in the feral tobacco, which might indicate a polyphyletic origin or early divergence from all cultivars examined. Overall, only in tobacco is there any evidence that NCI may have influenced genome evolution, and here further data are required to verify chromosome identity.