Ribosomal RNA Genes Evolution in Tragopogon: a Story of New and Old World Allotetraploids and the Synthetic Lines

Malinska & al. • Evolution of rDNA in Tragopogon TAXON 60 (2) • April 2011: 348–354

Ribosomal RNA genes evolution in Tragopogon: A story of New and Old World allotetraploids and the synthetic lines

Hana Malinska,1 Jennifer A. Tate,2 Evgeny Mavrodiev,2 Roman Matyasek,1 K. Yoong Lim,3 Andrew R. Leitch,3 Douglas E. Soltis,2 Pamela S. Soltis4 & Ales Kovarik1

1 Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i, Laboratory of Molecular Epigenetics, Královopolská 135, 61265 Brno, Czech Republic 2 Department of Botany, University of Florida, Gainesville, Florida 32611, U.S.A. 3 School of Biological Sciences, Queen Mary, University of London, E1 4NS, U.K. 4 Florida Museum of Natural History, University of Florida, Gainesville, Florida 32611, U.S.A. Authors for correspondence: Hana Malinska, [email protected] and Ales Kovarik, [email protected]

Abstract Tragopogon mirus Ownbey and Tragopogon miscellus Ownbey are recent allotetraploids that have formed recurrently within the last 80 years, following the introduction of the diploids T. dubius Scop., T. pratensis L. and T. porrifolius L. from Europe to North America. In some areas, the progenitor diploids still occur along with expanding populations of the allotetraploids, and the polyploids at those locations likely represent descendents of the nearby diploids. In most natural populations of T. mirus and T. miscellus, there are far fewer rDNA units of the common parent (T. dubius) than there are of the other diploid parent and in some rarely occurring individuals, one parental locus was >90% deleted. Nevertheless, in contrast to several ancient Old World allotetraploids, gene copies from both parents are readily detected by molecular methods in both T. mirus and T. miscellus. In one population of T. mirus and herbarium specimens collected at the time of species origin the gene ratios were balanced. Several lineages of T. mirus and T. miscellus were recently successfully resynthesized from diploid species. Among 181 synthetic individuals analyzed, we observed frequent deviations from copy-number additivity; that is in most cases, the T. dubius homeologs were reduced in copy number. At the epigenetic level, the genes of T. dubius origin dominate expression in most natural and synthetic allopolyploids. The fact that some rDNA genotypes seen in 80-year-old allopolyploids are already evident in the first generation of synthetic lines supports the hypothesis that the extent and tempo of rDNA homogenization in older allopolyploids is largely influenced by genetic and epigenetic changes in the early generations. Thus, T. mirus and T. miscellus that formed repeatedly in the wild within the last century represent a unique system to study the early stages of genome evolution following interspecific hybridization and genome duplication. This review summarizes recent works on the rDNA chromosomal organization, repeat and loci inheritance and gene expression.

Keywords allopolyploid; homogenization; nucleolar dominance; ribosomal DNA; Tragopogon

Introduction problems (Hadac, 1989; Ainouche & al., 2004). The success of newly formed angiosperm polyploids is partly attributable to In 1970, Susumu Ohno promoted the idea that gene and their highly plastic genome structure, as manifested by devia- genome duplications are the principle creative force in evolu- tions from Mendelian inheritance patterns of genetic loci and tion (Ohno, 1970). Perhaps no other group of living organisms chromosome aberrations (Leitch & Leitch, 2008). supports his hypothesis better than land plants. Chromosome Previous studies on natural and synthetic allopolyploids counts and other approaches suggest that between 30% and have shown that united parental genomes undergo active, fast, 100% of angiosperm species are polyploids (Masterson, 1994; and in some cases reproducible and directed processes of ge- Bennett, 2004; Soltis & al., 2009). Genomic studies have re- nome reorganization, probably due to intergenomic interactions vealed that all plant genomes sequenced to date have undergone (Soltis & Soltis, 1995). Reorganizations include both genetic one or more genome wide duplications in their evolutionary and epigenetic changes, which are observable after just a few history (Cui & al., 2006; Fawcett & al., 2009; Van de Peer & generations and even in F1 hybrids (e.g., Dadejova & al., 2007; al., 2009). Interspecific hybridization followed by multiplica- Kopecky & al., 2008; for review, see Chen & Ni, 2006). Most tion of parental chromosome sets is called allopolyploidy and genetic changes involve sequence loss or elimination often is believed to be a major driving force of angiosperm species associated with genome downsizing (Pestsova & al., 1998; evolution (Soltis & Soltis, 1995). Many economically important Kashkush & al., 2002), intergenomic chromosomal transloca- crops (e.g., wheat, oilseed rape, cotton, coffee, banana, tobacco) tions (Kenton & al., 1993), or the expansion of genome-specific are relatively recent allopolyploids (Leitch & Leitch, 2008). transposons (Zhao & al., 1998). Epigenetic silencing of parental The formation of allopolyploid species is, however, not always genes caused by the modification of chromatin is believed to beneficial since rapidly expanding populations of invasive allo reconcile regulatory incompatibilities between parental subge- polyploid weeds may cause severe ecological and economic nomes (Adams & Wendel, 2005). In contrast to the evolution of

348 TAXON 60 (2) • April 2011: 348–354 Malinska & al. • Evolution of rDNA in Tragopogon

single-copy genes, members of multigene families do not often 2008). Eukaryotes regulate the effective dosage of their rRNA evolve independently, but in a concerted manner. In multigene genes, expressing fewer than half of the genes at the same families, limited sequence variability is often observed among time (Neves & al., 2005). Likewise, genetic hybrids displaying individual copies. Despite this high intraspecific homogeneity, nucleolar dominance transcribe rRNA genes inherited from interspecific divergence is not affected. This phenomenon has one parent, but silence the other parental set (Volkov & al., been called concerted evolution (Arnheim & al., 1980). The 2007). Interplay of DNA methylation, histone modification, underlying molecular processes are unequal crossing over and and chromatin-remodeling activities establishes silencing in gene conversion. The degree of homogenization is thus a result rDNA loci (Preuss & Pikaard, 2007). of the interplay between homogenization processes and novel The evolution of rDNA has stimulated much research. Ex- base substitutions (Schlotterer & Tautz, 1994). pression patterns of rRNA genes have been studied at inter In plants, nuclear ribosomal DNA (rDNA) units oc- phase by observing the behavior of nucleoli (Leitch, 2000). cur in tandem arrays at one or several loci (for review see Epigenetic processes can lead to inactivation of entire rDNA Hemleben & Zentgraf, 1994). These rRNA genes, frequently arrays (nucleolar dominance, Honjo & Reeder, 1973; Dadejova used as phylogeny markers, are organized on chromosomes & al., 2007; Preuss & Pikaard, 2007). Sequences can undergo as long arrays of tandemly arranged units. Each large 35S concerted evolution involving sequence homogenization (Do- rDNA unit contains the 18S, 5.8S and 26S rRNA genes, the ver, 1982; Wendel & al., 1995). Finally, the sequence data from internal transcribed spacers (ITS), and the intergenic spacer the internally transcribed spacers (nuclear ribosomal ITS) are (IGS) (Fig. 1). The large polycistronic 35S transcript is endo- regularly used to construct phylogenetic trees (Nieto Feliner nucleotically processed to produce mature rRNA molecules. & Rossello, 2007; Poczai & Hyvonen, 2010). The 5S genes encoding 120 nt transcripts are usually, but not In this review we will summarize the data from previous always (Garcia & al., 2009), located at different chromosomal and ongoing studies in recently formed Tragopogon allotetra- loci than 35S rDNA. The genic regions are highly conserved ploids, focusing on rDNA evolution. even between distally related species, whereas the intra and intergenic regions are highly variable. For example, the sequence polymorphisms within IGS and ITS are sufficient to The origin of Tragopogon resolve species relationships within most genera (e.g., Lihova allotetraploids & al., 2004; for review, see Alvarez & Wendel, 2003). The IGS, which contains the transcriptional start site as well as Tragopogon (Asteraceae) comprises approximately 100– genetic and epigenetic features that influence the regulation 150 species native to Eurasia. Most of them are diploids (2n = of the downstream genes, diverges rapidly, and substantial 12), but at least twelve species are ancient polyploids (2n = 24, differences in structure may occur even within a species (Ben- 36, 56–58) with generally unknown parentages (Mavrodiev nett & Smith, 1991; Skalicka & al., 2003; Komarova & al., & al., 2008).

Fig. 1. The chromosomal and genomic structure of rDNA. Clusters of active rRNA genes are often located at the terminal part of the chromosome, which is cytogenetically revealed as a secondary constriction (upper part). Note, two decondensed domains (arrows) are flanked by knobs of highly condensed rDNA chromatin. Each cluster is composed of hundreds to thousands of units arranged head to tail in tandem. Each unit is composed of conserved genic regions encoding 18S-5.8S-26S rRNA that are separated by the internally transcribed spacers (ITS1, ITS2) and the intergenic spacer (IGS). IGS contains an RNA polymerase I promoter and other regulatory elements. The unit is transcribed into a polycistronic 18S-5.8S-26S rRNA precursor (pre-rRNA) that is subjected to spacer excision and maturation into functional structural RNA molecules.

349 Malinska & al. • Evolution of rDNA in Tragopogon TAXON 60 (2) • April 2011: 348–354

Three diploid species, Tragopogon dubius Scop., Trago- 1920s (the earliest collection of T. dubius, the common diploid pogon porrifolius L. and Tragopogon pratensis L., were in- parent of both tetraploid species, is from 1928; Ownbey, 1950), troduced into the Palouse region of eastern Washington and so we assume that T. mirus and T. miscellus cannot be more adjacent Idaho, U.S.A., in the early 1900s (Ownbey, 1950). The than ca. 80 years old. According to the geographic distribution, introduction of these diploid species into the Palouse brought it is estimated that there may be as many as 21 separate origins them into close contact, which rarely occurs in the Old World. of T. miscellus and 12 of T. mirus (Cook & al., 1998). We are Using morphology and cytology, Ownbey (1950) demonstrated still identifying new natural populations of allotetraploids so it that Tragopogon mirus Ownbey and Tragopogon miscellus is likely that the speciation process is far from being finished. Ownbey are allotetraploids (2n = 24) whose diploid parents (2n = 12) are T. dubius and T. porrifolius and T. dubius and T. pratensis, respectively (Fig. 2). The ancestries of both tetraploids Ribosomal DNA (rDNA) in natural were subsequently confirmed through flavonoid, isozyme, and populations of T. mirus and DNA studies (for review see Soltis & al., 2004). The three dip- T. miscellus loids probably did not occur together in the Palouse prior to the In most populations of T. mirus and T. miscellus examined, there are far fewer 18S-5.8S-26S rDNA units of T. dubius than of the other diploid parent (Fig. 3, green columns). This situation is typical since the similar trend was observed in nine out of eleven populations of allotetraploids of independent origin (Kovarik & al., 2005; Koh & al., 2010). This population-level analysis revealed relatively low intrapopula- tion heterogeneity in parental gene ratios. Nevertheless, some populations contained rare individuals that markedly differed from the rest of populations by the extent of gene losses. For example, reduction of T. dubius genes in one individual (33A) of T. mirus from Rosalia (2603) population (Fig. 4) was dra- matic and the Southern blot hybridization patterns resembled monomorphic genotypes (Kovarik & al., 2005) often seen in relatively ancient allopolyploids (Wendel & al., 1995; Joly & al., 2004; Kovarik & al., 2004). Thus, it seems that individuals displaying nearly homogenized rDNA arrays may occur in low frequency within allopolyploid populations of Tragopogon, which typically comprise hundreds to thousands of individu- Fig. 2. Evolutionary relationships between allotetraploid and diploid als. It is necessary to mention that two populations of T. mirus Tragopogon species. Arrows indicate the direction of interspecific hybridization. In T. mirus, all of the recurrent origins involve T. dubi- from Albion (not shown) and Palouse (Fig. 3, population 2602) us as paternal plant and T. porrifolius as maternal parent. Tragopogon displayed balanced homeolog gene ratios indicating slower miscellus has formed reciprocally, but most of the recurrent origins tempo of elimination or large copy-number misbalances be- involve T. dubius as the paternal parent. tween parental diploids. This issue was addressed by the rDNA

Fig. 3. Inheritance of parental rDNAs and their expression patterns in natural populations of Tragopogon mirus and T. miscellus (data from Matyasek & al., 2007). X-axis: population numbers, each population represents locality of origin. Y-axis: proportion of T. dubius units. The rDNA ratios were determined by Southern blot hybridization (Kovarik & al., 2005). Expression of homeologous genes was analyzed by cDNA CAPS (Matyasek & al., 2007). We identified populations with no (population 2602), partial (populations 2604, 2605) and complete (populations 2601, 2603, 2606) nucleolar dominance.

350 TAXON 60 (2) • April 2011: 348–354 Malinska & al. • Evolution of rDNA in Tragopogon

analysis in synthetic lines (see further below). We observe simi- there is divergence in locus numbers between the homeologs larly altered ITS ratios among reciprocally formed individuals (Lim & al., 2008). of T. miscellus (populations 2604 and 2605). This suggests In conclusion, molecular and cytogenetic studies show that the direction of rDNA fixation in Tragopogon does not that there may be more than one pathway leading to the non- seem to be determined by the direction of the cross and that additive patterns of rDNA inheritance in Tragopogon allo nuclear-cytoplasmic interactions (Gill, 1991; Kovarik & al., tetraploids: (1) elimination or amplification of repeats within 2005; Matyasek & al., 2007) may not play a significant role an array can occur without changes in locus number (as in the in homogenization of parental rDNAs in these allotetraploids. case of 18S-5.8S-26S rDNA locus); (2) a change in the number In contrast, structural and epigenetic features of loci could be of rDNA-bearing chromosomes (and loci) without any change governing the tempo of intra and interlocus homogenization. in the number of genes at each locus (as with the 5S rDNA From molecular studies (Southern blot and genomic CAPS locus); or (3) a combination of both, although this situation has [Cleaved Amplified Polymorphic Sites]) of the Tragopogon not yet been detected. allopolyploids, it is not clear whether the non-additive pattern of inheritance observed is caused by a decrease in copy number (elimination) or unit replacement (e.g., via homogeniza- Herbarium specimens tion mechanisms). Combined fluorescent and genomic in situ hybridization analyses (FISH/GISH) have recently enabled Nearly equal numbers of parental rDNA types are found the genomic origin of the allopolyploid chromosomes to be in the DNA samples isolated from herbarium specimens of determined (Fig. 4 and Lim & al., 2008). In plant 2603-33A, both allotetraploids collected in 1949 and 1953 (Kovarik & al., there is a drastic reduction in the size of the 18S-5.8S-26S 2005). It is clear that these Marion Ownbey’s early collections rDNA locus on both A chromosomes of T. dubius origin as of T. mirus and T. miscellus are very close to the time of origin compared to the parental diploids, while the loci on the home- of these newly formed species (see Ownbey, 1950). The popula- ologous chromosomes seem to be unaffected. Thus, it seems tions described by Ownbey probably formed in the early 1940s, that there have been uniparental deletions of units rather than approximately 60 years ago. Given that these plants are bienni- interlocus homogenization in Tragopogon. Interestingly, size als, the changes seen in today’s individuals must have occurred reductions affect the rDNA loci on both of the T. dubius ho- within the past 30 generations. Our sampling of herbarium mologues, suggesting that deletions are homozygous and per- specimens is rather low, so it is not possible to make definitive haps well stabilized, as apparent from analysis of the progeny conclusions about the speed of the homogenization process. (not shown). In addition to the identification of the parental However, relatively balanced ratios of parental ITS types are origin of rDNA loci, the cytogenetic analysis reveals another also found in one population of current natural allotetraploids unexpected feature. Although numbers of chromosomes of (population 2602, Fig. 3), which may represent a very recently T. mirus and T. miscellus appear intact (2n = 4x = 24), there formed population of T. mirus or rDNA genotype stabilized by are frequent chromosomal imbalances caused by subgenomic unknown mechanism. monosomies compensed by trisomies (Lim & al., 2008). For example, five of the eight T. mirus karyotypes and two of the three T. miscellus karyotypes with 2n = 24 chromosomes Old World allotetraploids have unbalanced genomic contributions. These individuals were randomly chosen suggesting that karyotypic variation Several Tragopogon allotetraploids 2n = 24 occur in West may occur relatively frequently within the populations. We Asia and Southern Europe. In contrast to New World allo further demonstrated that imbalances mentioned above also tetraploids, the species are largely allopatric, and populations contribute to copy-number alterations of 5S rDNA because grow isolated from their putative parents indicating the ancient

Fig. 4. Karyotype analysis of two sister individuals of T. mirus using FISH/GISH. Plant 2603-33A has a homozygous macrodele- tion of a T. dubius 18S-5.8S-26S rDNA locus (hash), as well as trisomy and monosomy of T. dubius (D-genome) and T. porrifolius (P-genome) genetic compartments, respectively (underlined). Plant 2603-33B has rDNA signals typical for a given population 2603 (adapted from Lim & al., 2008). Secondary constrictions are marked with arrows.

351 Malinska & al. • Evolution of rDNA in Tragopogon TAXON 60 (2) • April 2011: 348–354

origin of most Old World allotetraploids (Ownbey, 1950). and expression of rRNA genes (Malinska & al., 2010). At the Mavrodiev and colleagues (2008) recently studied the distribu- molecular level, we observed considerable variation in rRNA tion of nuclear rDNA markers in an effort to decipher parentage gene copy numbers, not only across lineages, but also among of several ancient allotetraploids. They carried out sequencing individuals obtained from the same cross. The frequent changes of ETS (external transcribed spacer) and ITS subregions of the involved rapid elimination of T. dubius–derived rDNA units, rDNA unit in Tragopogon buphthalmoides, T. castellanus, T. col- a scenario that is reminiscent to that observed in most natural oratus, T. gracilis, T. latifolius, and T. tuberosus (all, 2n = 24). allotetraploids. We concluded that some patterns of rDNA evo- Only parentage of T. castellanus could be successfully deter- lution in Tragopogon allotetraploids could be experimentally mined based on retention of ribotypes corresponding to putative reproduced (at least to some extent). The rapid modifications of diploid progenitors close to modern T. crocifolius and T. lamot- rDNA loci may be controlled by a gene-loss mechanism induced tei. Yet, the rDNA ratios were highly skewed and the low-copy by allopolyploid stress, which is presently poorly understood ribotypes were only revealed after extensive cloning analysis. (Song & al., 1995; Shaked & al., 2001; Skalicka & al., 2005). It Similarly, two variants were obtained from cloning of ITS vari- is necessary to mention that in synthetic lines, we also observed ants from T. tuberosus. On the other hand T. buphthalmoides changes that were never detected in natural populations. Perhaps, and T. gracilis retained only one parental ITS type. T. latifo- some genotypes arising in early generation of allopolyploids lius was interesting in that two samples, one collected from might be later eliminated by natural selection. In this context, Armenia (81639) and a second from the other Asian population synthetic allotetraploid lines display variable degrees of fertil- (1898), differed in the homogenity of ITS sequences. The sample ity and viability. Translocations, deletions, chromosome loss, from the Armenian population displayed a uniform sequencing and quadrivalent formation in meiosis, have been observed in electropherogram while the sample from Asia showed several some individuals, indicating meiotic instability in these early- polymorphic sites. One important conclusion from these stud- generations allopolyploids (Lim & al., 2008). ies is that ITS homogenization may proceed at different rates in different species, and that the age is probably not the only factor influencing retention of parental genes in unified nucleus. Iden- Epigenetic aspects of rDNA tification of the origin of ancient allopolyploids could be masked evolution by several factors: (1) the diploid genome donors may go extinct; (2) there was uneven contribution of copy number of the rRNA Besides homogenization of parental rDNA units, another genes from genome donors; (3) finally, the allopolyploids could interesting aspect of ribosomal RNA biology is the epigenetic have already evolved novel species-specific types eliminating silencing of the thousands of units within rDNA loci (Preuss parental types. This hypothesis is supported by the analysis of & Pikaard, 2007). At the cytogenetic level, this phenomenon Euroasian hybrid, T. pratensis × T. porrifolius that possesses an was discovered in the 1930s by studies of interspecific hybrids extremely reduced genome (Krahulec & al., 2005), and in which of Crepis (Navashin, 1934). The transcriptional silencing of no parental ITS types were identified (Kovarik, unpub. results). one parental gene family is called nucleolar dominance and is seen in many allopolyploids and synthetic hybrids (Volkov & al., 2007). We have previously examined expression of home- Synthetic hybrids and ologous rRNA genes by cDNA CAPS and nuclease protection allopolyploids assay (Matyasek & al., 2007). In natural Tragopogon allotetraploids, we identified one population without nucleolar domi- The variation observed among natural polyploid individu- nance, two populations with partial nucleolar dominance, and als can be explained either by genetic and/or epigenetic changes three populations with complete nucleolar dominance (Fig. 3, within polyploid individuals or by the presence of variation in red columns). Unexpectedly, populations with a reduced num- parental plants. The parental species can differ in the number of ber of T. dubius copies usually show nearly complete suppres- rDNA units present, so the tetraploid progeny can vary depend- sion of the second genome donor, suggesting that there is an ing on combination of parents which are used for hybridization. inverse correlation between gene copies and their expression Changes in newly established polyploid genomes can also occur capacity. In T. mirus, we observe high or low variability (de- as a result of the stress imposed when divergent genomes come pending on population, Fig. 3) in nucleolar dominance among together in the same nucleus (Song, 1995; Leitch & Leitch, 2008). individuals within a given population, as well as among differ- These principal questions can be best addressed by analyzing ent populations, suggesting that the type of dominance cannot synthetic lines, whose genetic parents are well defined. How- be determined so easily. For example in population 2602, which ever, in many systems, the generation of synthetic allopolyploid has a balanced copy-number of both rDNA parental units, the lines has been unsuccessful due to physiological and genetic majority of polyploids show co-dominance of both parents, incompatibility of the hybridizing species (Allen & al., 1983; but individuals with nearly complete T. dubius or T. porrifolius Lim & al., 2008). In Tragopogon, resynthesis of allotetraploids dominance can be found with frequency of 3% or 14% respec- from the diploid species T. dubius, T. porrifolius and T. praten- tively (Fig. 3 and Matyasek & al., 2007). sis has been successful (Tate & al., 2009), and seven lines of At the cytogenetic level, we identify secondary constric- T. mirus and four lines of T. miscellus have been obtained. In tions, a hallmark of transcriptional activity (McClintock, 1934), this material (181 individuals), we have analyzed inheritance on chromosomes of T. dubius origin in both tetraploid species

352 TAXON 60 (2) • April 2011: 348–354 Malinska & al. • Evolution of rDNA in Tragopogon

(Fig. 4 and Lim & al., 2008). Secondary constrictions do not Literature cited occur in the T. porrifolius and T. pratensis rDNA loci, except of Abbott, R.J. & Lowe, A.J. 2004. Origins, establishment and evolution populations with codominant rDNA phenotype. Decondensa- of new polyploid species: Senecio cambrensis and S. eboracensis tion of the T. porrifolius locus was also observed in one plant in the British Isles. Biol. J. Linn. Soc. 82: 467–474. (33A) in which the T. dubius rDNA copy-number was extremely Adams, K.L. & Wendel, J.F. 2005. Polyploidy and genome evolution reduced to 5% (Fig. 4, 33A). Interestingly, this individual lost in plants. Curr. Opin. Pl. Biol. 8: 135–141. transcriptional dominance in rapidly dividing tissues such Ainouche, M.L., Baumel, A., Salmon, A. & Hubbard, C.E 2004. as roots and flowers, while in leaf tissue, the dominance of Spartina anglica Schreb.: A natural model system for analysing early evolutionary changes that affect allopolyploid genomes. Biol. T. dubius genes (typical phenotype for a given population) was J. Linn. Soc. 82: 475–474. retained (Malinska & al., 2010). We speculate, that stability of Allen, G.A., Dean, M.L. & Chambers, K.L. 1983. Hybridization stud- rRNA silencing may depend on the copy number of genes in the ies in the Aster occidentalis (Asteraceae) polyploid complex of epigenetically favorable loci, that is the one of T. dubius origin. Western North America. Brittonia 35: 353–361. Alvarez, I. & Wendel, J.W. 2003. Ribosomal ITS sequences and plant phlyogenetic inference. Molec. Phylog. Evol. 29: 417–434. Arnheim, N., Krystal, M., Schmickel, R., Wilson, G., Ryder, O. Future directions for research & Zimmer, E. 1980. Molecular evidence for genetic exchanges among ribosomal genes on non-homologous chromosomes in man Although many excellent studies have shown rapid chro- and apes. Proc. Natl. Acad. Sci. U.S.A. 77: 7323–7327. mosomal and sequence evolution in allopolyploids, it remains Bennett, M.D. 2004. Perspectives on polyploidy in plants—ancient and to be determined whether these changes are stochastic or di- neo. Biol. J. Linn. Soc. 82: 411–423. rected, random or reproducible. Analysis of synthetic hybrids Bennett, R.I., & Smith, A.G. 1991. Use of a genomic clone for ribosomal RNA from Brassica oleracea in RFLP analysis of Brassica and polyploids could, in part, address this tantalizing issue. species. Pl. Molec. Biol. 16: 685–688. In our initial study of rDNA inheritance (Kovarik & al., Chen, Z.J. & Ni, Z.F. 2006. Mechanisms of genomic rearrangements 2005), we analyzed 10–20 tetraploid individuals per population. and gene expression changes in plant polyploids. Bioessays 28: However, there may be hundreds to thousands of individuals 240–252. within a population. Given the relative simplicity of the meth- Cook, L.M., Soltis, P.S., Brunsfeld, S.J. & Soltis, D.E. 1998. Multiple independent formations of Tragopogon tetraploids (Asteraceae): ods used, it may be feasible to screen most individuals in a Evidence from RAPD markers. Molec. Ecol. 7: 1293–1302. given locality across generations. Such an approach would give Cui, L., Wall, P.K., Leebens-Mack, J.H., Lindsay, B.G., Soltis, D.E., a comprehensive view of genetic variation and the evolutionary Doyle, J.J., Soltis, P.S., Carlson, J.E., Arumuganathan, K. & trends governing the dynamics of natural populations. Barakat, A. 2006. Widespread genome duplications throughout It will be interesting to compare patterns of rRNA gene the history of flowering plants. Genome Res. 16: 738–749. evolution in other recently formed allopolyploids. For example, Dadejova, M., Lim, K.Y., Souckova-Skalicka, K., Matyasek, R., Grandbastien, M.A., Leitch, A. & Kovarik, A. 2007. Transcrip- the ca. 300 years old Cardamine insueta and C. schulzii al- tion activity of rRNA genes correlates with a tendency towards ready show evidence for intergenomic recombination of ITS intergenomic homogenization in Nicotiana allotetraploids. New (Urbanska & al., 1997). Spartina anglica and Senecio camb- Phytol. 174: 658–668. rensis, allopolyploids that have formed <150 years ago (Abbott Dover, G.A. 1982. Molecular drive: A cohesive mode of species evolu- & Lowe, 2004; Ainouche & al., 2004) are candidates for pop- tion. Nature 299: 111–117. ulation-level studies of rDNA. Fawcett, J., Maere, S. & Van de Peer, Y. 2009. Plants with double genomes might have had a better chance to survive the Creta- In allopolyploids, genes inherited from different parents ceous-Tertiary extinction event. Proc. Natl. Acad. Sci. U.S.A. 106: can be differentially expressed in different tissues (a phenom- 5737–42. enon known as subfunctionalization; Adams & Wendel, 2005). Garcia, S., Lim, K.Y., Chester, M., Garnatje, T., Pellicer, J., Valles, It will be interesting to determine the developmental stability J., Leitch, A.R., & Kovarik, A. 2009. Linkage of 35S and 5S of rRNA silencing. rRNA genes in Artemisia (family Asteraceae): First evidence from angiosperms. Chromosoma 118: 85–97. It is known from Arabidopsis models that rRNA gene Gill, B.S. 1991. Nucleocytoplasmic interaction (NCI) hypothesis of silencing is dependent on cytosine and histone methylation, genome evolution and speciation in polyploid plants. Pp. 48–53 histone acetylation and siRNA (Preuss & Pikaard, 2007). It will in: Sasakuma, T. & Kinoshita, T. (eds.), Proceedings of the Kihara be interesting to determine which of these epigenetic factors Memorial International Symposium on Cytoplasmic Engineering predispose the T. dubius rDNA to transcriptional dominance in Wheat. Yokohama: Kihara Memorial Foundation. in the Tragopogon allotetraploids. Hadac, E. 1989. Ecological significance of polyploidy in high mountain plants and plant communities. Folia Geobot. 24: 51–56. Hemleben, V. & Zentgraf, U. 1994. Structural organisation and regulation of transcription by RNA polymerase I of plant nuclear ribo- Acknowledgements somal genes. Pp. 3–24 in: Nover, L. (ed.), Results and problems in cell differentiation, vol. 20, Plant promoters and transcription The work was supported by NERC (U.K.), Scientific Foundation factors. Berlin, Heidelberg: Springer. Honjo, T. & Reeder, R.H. 1973. Preferential transcription of Xenopus of the Czech Republic. This research was funded by the Grant Agency laevis ribosomal RNA in interspecies hybrids between Xenopus of the Czech Republic (204/09/H002, P501/10/0208, 206/09/1751) and laevis and Xenopus muller. J. Molec. Biol. 80: 217–228. the Academy of Sciences of the Czech Republic (AVOZ50040507 and Joly, S., Rauscher, J.T., Sherman-Broyles, S.L., Brown, A.H. AVOZ50040702). & Doyle, J.J. 2004. Evolutionary dynamics and preferential

353 Malinska & al. • Evolution of rDNA in Tragopogon TAXON 60 (2) • April 2011: 348–354

expression of homeologous 18S-5.8S-26S nuclear ribosomal genes Neves, N., Delgado, M, Silva, M., Caperta, A., Morais-Cecilio, L. in natural and artificial glycine allopolyploids. Molec. Biol. Evol. & Viegas, W. 2005. Ribosomal DNA heterochromatin in plants. 21: 1409–1421. Cytogenet. Genome Res. 109: 104–111. Kashkush, K., Feldman, M. & Levy, A.A. 2002. Gene loss, silencing Nieto Feliner, G., & Rossello, J.A. 2007. Better the devil you know? and activation in a newly synthesized wheat allotetraploid. Genet- Guidelines for insightful utilization of nrDNA ITS in species-level ics 160: 1651–1659. evolutionary studies in plants. Molec. Phylog. Evol. 44: 911–919. Kenton, A., Parokonny, A.S., Gleba, Y.Y. & Bennett, M.D. 1993. Ohno, S. 1970. Evolution by gene duplication. New York: Springer. Characterization of the Nicotiana tabacum L. genome by molecular Ownbey, M. 1950. Natural hybridization and amphiploidy in the genus cytogenetics. Molec. Gen. Genet. 240: 159–169. Tragopogon. Amer. J. Bot. 37: 487–499. Koh, J., Soltis, P.S. & Soltis, D.E. 2010. Homeolog loss and expres- Pestsova, E.G., Goncharov, N.P. & Salina, E.A. 1998. Elimination of sion changes in natural populations of the recently and repeatedly a tandem repeat of telomeric heterochromatin during the evolution formed allotetraploid Tragopogon mirus (Asteraceae). BMC Ge- of wheat. Theor. Appl. Genet. 97: 1380–1386. nomics 11: 92. DOI: 10.1186/1471-2164-11-97. Poczai, P. & Hyvonen, J. 2010. Nuclear ribosomal spacer regions in Komarova, N.Y., Grimm, G.W., Hemleben, V. & Volkov, R.A. 2008. plant phylogenetics: Problems and prospects. Molec. Biol. Rep. Molecular evolution of 35S rDNA and taxonomic status of Lyco- 37: 1897–1912 persicon within Solanum sect. Petota. Pl. Syst. Evol. 276: 59–71. Preuss, S. & Pikaard, C.S. 2007. rRNA gene silencing and nucleolar Kopecky, D., Lukaszewski, A.J. & Dolezel, J. 2008. Cytogenetics dominance: Insights into a chromosome-scale epigenetic on/off of Festulolium (Festuca × Lolium hybrids). Cytogenet. Genome switch. Biochem. Biophys. Acta 1769: 383–392. Res. 120: 370–383 Schlotterer, C. & Tautz, D. 1994. Chromosomal homogeneity of Dro- Kovarik, A., Matyasek, R., Lim, K.Y., Skalicka, K., Koukalova, B., sophila ribosomal DNA arrays suggests intrachromosomal exchan- Knapp, S., Chase, M. & Leitch, A. 2004. Concerted evolution ges drive concerted evolution. Curr. Biol. 9: 777–783. of 18-5.8-26S rDNA repeats in Nicotiana allotetraploids. Biol. J. Shaked, H., Kashkush, K., Ozkan, H., Feldman, M. & Levy, A.A. Linn. Soc. 82: 615–625. 2001. Sequence elimination and cytosine methylation are rapid and Kovarik, A., Pires, J.C., Leitch, A.R., Lim, K.Y., Sherwood, A.M., reproducible responses of the genome to wide hybridization and Matyasek, R., Rocca, J., Soltis, D.E. & Soltis, P.S. 2005. Rapid allopolyploidy in wheat. Pl. Cell 13: 1749–1759. concerted evolution of nuclear ribosomal DNA in two Tragop- Skalicka, K., Lim, K.Y., Matyasek, R., Koukalova, B., Leitch, A. & ogon allopolyploids of recent and recurrent origin. Genetics 169: Kovarik, A. 2003. Rapid evolution of parental rDNA in a synthetic 931–944. tobacco allotetraploid line. Amer. J. Bot. 90: 988–996. Krahulec, F., Novak, J. & Kaplan, Z. 2005. Tragopogon porrifolius Skalicka, K., Lim, K.Y., Matyasek, R., Matzke, M., Leitch, A.R. & × T. pratensis: The present state of an old hybrid population in Kovarik, A. 2005. Preferential elimination of repeated DNA se- Central Bohemia, the Czech Republic. Preslia 77: 297–306 quences from the paternal Nicotiana tomentosiformis genome do- Leitch, A.R. 2000. Higher levels of organization in the interphase nu- nor of a synthetic allotetraploid tobacco. New Phytol. 166: 291–303 cleus of cycling and differentiated cells. Microbiol. Molec. Biol. Soltis, D.E., Albert, V.A., Leebens-Mack, J., Bell, Ch.D., Paterson, Rev. 64: 138–152. A.H., Zheng, Ch., Sankoff, D., dePamphilis, C.W., Wall, P.K. Leitch, A.R. & Leitch, I.J. 2008. Genomic plasticity and the diversity & Soltis, P.S. 2009. Polyploidy and angiosperm diversification. of polyploid plants. Science 320: 481–483. Amer. J. Bot. 96: 336–348. Lihova, J., Aguilar, J.F., Marhold, K. & Feliner, G.R. 2004. Origin Soltis, D.E. & Soltis, P.S. 1995. The dynamic nature of polyploid ge- of the disjunct tetraploid Cardamine amporitana (Brassicaceae) nomes. Proc. Natl. Acad. Sci. U.S.A. 92: 8089–8091. assessed with nuclear and chloroplast DNA sequence data. Amer. Soltis, D.E., Soltis, P.S., Pires, J.C., Kovarik, A. & Tate, J. 2004. J. Bot. 91: 1231–1242 Recent and recurrent polyploidy in Tragopogon (Asteraceae): Ge- Lim, K.Y., Soltis, D.E., Soltis, P.S., Tate, J., Matyasek, R., Srubarova, netics, genomic, and cytogenetic comparisons. Biol. J. Linn. Soc. H., Kovarik, A., Pires, J.C., Xiong, Z. & Leitch, A.R. 2008. 82: 485–501. Rapid chromosome evolution in recently formed polyploids in Song, K., Lu, P., Tang, K. & Osborn, T.C. 1995. Rapid genome change Tragopogon (Asteraceae). PLoS ONE 3: e3353. DOI: 10.1371/jour- in synthetic polyploids of Brassica and its implications for poly- nal.pone.0003353. ploid evolution. Proc. Natl. Acad. Sci. U.S.A. 92: 7719–7723. Malinska, H., Tate, J.A., Matyasek, R., Leitch, A.R., Soltis, D.E., Tate, J.A., Symonds, V., Doust, A., Buggs, R., Mavrodiev, E., Soltis, P.S. & Kovarik, A. 2010. Similar patterns of rDNA evolu- Majure, L., Soltis, P.S. & Soltis, D.E. 2009. Synthetic polyploids tion in synthetic and recently formed natural populations of Trago- of Tragopogon miscellus and T. mirus (Asteraceae): 60 years after pogon (Asteraceae) allotetraploids. BMC Evol. Biol. 10: 291. DOI: Ownbey’s discovery. Amer. J. Bot. 96: 979–988. 10.1186/1471-2148-10-291. Urbanska, K.M., Hurka, H., Landolt, E., Neuffer, E. & Mummen- Masterson, J. 1994. Stomatal size in fossil plants: Evidence for poly- hoff, K. 1997. Hybridization and evolution in Cardamine (Bras- ploidy in majority of angiosperms. Science 264: 421–424. sicaceae) at Urnerboden, Central Schwitzerland: Biosystematics Matyasek, R., Tate, J.A., Lim, K.Y., Srubarova, H., Leitch, A.R., and molecular evidence. Pl. Syst. Evol. 204: 233–256. Soltis, D., Soltis, P. & Kovarik, A. 2007. Concerted evolution in Van de Peer, Y., Fawcett, J., Proost, S., Sterck, L. & Vandepoele, recently formed Tragopogon allotetraploids is typically associated K. 2009. The flowering world: A tale of duplications. Trends Pl. with an inverse correlation between gene copy number and expres- Sci. 14: 680–8. sion. Gentetics 176: 2509–2519. Volkov, R.A., Komarova, N.Y. & Hemleben, V. 2007. Ribosomal Mavrodiev, E., Soltis, P.S. & Solits, D.E. 2008. Putative parentage of DNA in plant hybrids: Inheritance, rearrangement, expression. six Old World polyploids in Tragopogon L. (Asteraceae: Scorzo- Syst. Biodivers. 5: 261–276. nerinae) based on ITS, ETS, and plastid sequence data. Taxon 57: Wendel, J.F., Schnabel, A. & Seelanan, T. 1995. Bidirectional inter- 1215–1232. locus concerted evolution following allopolyploid speciation in McClintock, B. 1934. The relationship of a particular chromosomal cotton (Gossypium). Proc. Natl. Acad. Sci. U.S.A. 92: 280–284. element to the development of the nucleoli in Zea mays. Z. Zellf. Zhao, X.P., Si, Y., Hanson, R.E., Crane, C.F., Price, H.J., Stelly, Mikroskop. Anat. 21: 294–328. D.M., Wendel, J.F. & Paterson, A.H. 1998. Dispersed repetitive Navashin, M. 1934. Chromosomal alterations caused by hybridization DNA has spread to new genomes since polyploid formation in and their bearing upon certain general genetic problems. Cytologia cotton. Genome Res. 8: 479–492. 5: 169–203.

354